Cell culture dish suitable for in situ electroporation and inducing desired cell potency and other behaviors

ABSTRACT

A cell culture dish made of non media-permeable material and having a base and a plurality of separate juxtaposed side-by-side wells having common interior well walls preventing physical contact or movement of chemical factors between the separated cell or tissue cultures, the walls having different heights and defining an outer “surround” cell or tissue culture and one or more inner “center” cell or tissue cultures to enable contained cell or tissue communication between the well spaces, the wells configured to comprise two or more cell or tissue cultures, the wells permitting signal communication between the cells or tissues situated within said wells, and further comprising wall material, electrode contacts electrodes and/or electrode contacts, and well dimensions suitable for facilitating electroporation. The inventor observed 100% efficiency in cell reprogramming with protein electroporation-orders of magnitude above other reported methods.

BACKGROUND OF THE INVENTION

The present invention relates to a novel culturing apparatus (assembly)which can be termed a cell culture dish. More particularly, the presentinvention is a “combined cell culture dish” or “dish-in-dish” apparatuscomprising at least one smaller cell culture dish fixedly positionedwithin a larger cell culture dish, and the number of such fixated cellculture dishes can include a multiple number of fixated cell culturedishes within one another, either concentric or eccentric, in any numberof geometric shapes, and without limitation to the number of petridishes included. An alternate embodiment of this invention can include aplurality of cell culture dishes juxtaposed side-by-side having commoninterior well walls, and the well walls may or may not be different inheight depending on the application. The combined cell culture dishdiffers from the prior art because the walls of said combined petridishes may be of different heights and made from any combination oftransparent and non-transparent materials that will allow juxtaposingand different cultures to grow simultaneously. Such separate butjuxtaposing culture growth can then be studied to determine whethercertain cultures grown separately and in close proximity influence eachother in certain ways. The combined cell culture dish of the presentinvention may or may not be fitted with single or multiple covers andmay or may not be stacked.

Examples of prior petri dishes may be found in the following U.S. Pat.No. 4,675,298 (Brusewitz, Gerhard). U.S. Pat. No. 4,160,700 (Boomus,Mary), and U.S. Pat. No. 3,660,243 (Young, Cecil).

The transcendent challenge for medicine in the 21st century will bereplacing damaged, worn-out or genetically-compromised cells.Transcription factors binding specifically to DNA play a vital role inregulating gene expression. It is the particular complement oftranscription factors within an individual cell, that determine whichcellular programs are active and which are turned off. In this capacitytranscription factors play a decisive role in determining andmaintaining cellular identity, as well as determining cellularvulnerability.

SUMMARY OF THE INVENTION

The present invention is directed to a novel cell culture dish having amulti-chambered construction which facilitates juxtaposition ofdifferent physically separated cultures. Said novel multi-chambered cellculture dish will permit co-culturing of any two or more separatecultures, whether those cultures are species related or not.

In general, the cell culture dish of the present invention comprises twoor more dishes which create a central compartment and one or moreperipheral compartments which surround the central compartment. Saidcentral and peripheral compartments may take the form of any shape,including, but not limited to. cylindrical, square, pentagonal, orhexagonal. The material used to construct said petri dish may include,but may not be limited to. any non media-permeable form of glass,plastic or metal or combination thereof, which will sustain culturegrowth and permit observation and recording of said culture growth,including, but not limited to. the recording of signal transduction.Separated areas created by utilizing the central compartment and one ormore peripheral compartments may be geometrically concentric oreccentric.

The petri dish of the present invention may comprise one or more disheswithin a dish or may be constructed of a single dish with a flat wellbottom having one or more sets of walls that extend from said wellbottom forming one or more separate enclosures having the same geometricshape or a variety of geometric shapes. The wall or walls areconstructed to ensure physical isolation of two or more sets of cellsfrom one another to prevent physical contact between the separated cellsor movement of chemical factors originating in the media or within thecells. The separate wells may or may not be numbered to enhance theidentification of certain cell cultures.

A preferred embodiment of this invention is depicted in FIG. 1 below.

The petri dish described above can be sterilized using either wet or dryheat. However, the petri dish may be a single use device as well. Theouter wall of the multi-chambered petri dish can be sized appropriatelyto fit high-perform an c e incubation and perfusion chambers for livecell imaging and to withstand temperatures ranging from 5 degrees belowCelsius to 50 degrees above Celsius. However, the multi-chambered petridishes of this invention may also withstand a host of temperaturesoutside the previously stated range.

One object of the present invention is to provide a multi-chambered cellor tissue culture dish suitable for assessing cell communication that isnot prohibited by intervention of the chamber walls.

Another object of this invention is to provide a cell or tissue culturedish having a plurality of separate wells which permit communicationbetween cells or tissues situated within said wells of signals orcommunication which might emanate from said cells or tissues.

Still another object of this invention is to provide a multi-chamberedcell or tissue culture dish with a transparent and flat bottom to enableconvenient and accurate viewing and analysis of the contents of eachseparate chamber.

A further object of this invention is to provide a multi-chambered cellor tissue culture dish which can be made from a number of transparentmaterials, including, but not limited to. glass, acrylic polymers,fluorinated ethylene propylene, ultra high molecular weightpolyethylene, polycarbonate, polystyrene, or any amorphoushigh-performance polymer, with or without electrodes and/or electrodecontacts facilitating electroporation.

Yet another object of this invention is to provide a multi-chamberedcell or tissue culture dish manufactured with well walls havingdifferent heights and defining an outer “surround” cell culture and oneor more inner “center” cell cultures to enable contained cell or tissuecommunication within the well spaces. Such communication would include,but would not be limited to. putative nociceptive cell signaling inphysically disconnected but proximal cell populations, includingcell-to-cell communications which are taking place after eliminating theavailability of any potential pathways for neural or diffusible factormediated cell-cell communication.

The ability to derive proliferating, self-renewing, multipotent andpluripotent cell population(s) from otherwise non-pluripotent, non-selfrenewing cells may have significant positive implications for all fieldsutilizing cellular therapies. These fields include bone marrowtransplantation, transfusion medicine, and gene therapy and enable theproduction of patient-specific stem cells and other desired cell types.Likewise, the ability to initiate differentiation of cells into neural,muscle, and various other desirable stem and somatic cell populations isand will also be of significant value to medicine and commercialprocesses involving animals. Accordingly, the present invention providesmethods for genetic production and uses of self-renewing cellpopulations, totipotent cell populations, multipotent cell populations,pluripotent cell populations, and differentiating/differentiated cellpopulations, e.g. neuronal cell populations, muscle cell populations,hematopoietic cell populations, etc., and other desired cell populationssuch as, for example, HIV resistant cell populations.

It is a proposition of the present invention that the efficientintroduction or overexpression of specific transcription factors, aloneor in combination with other cell fate determinants (e.g. notch, numb,numblike and other proteins, as well as certain miRNAs and othernon-coding RNAs), enables the interconversion of what have beenconsidered transitory (multipotent, pluripotent, and/or self-renewing)or fixed (differentiated or somatic) cellular phenotypes. The ability toreliably induce phenotypic conversion or cellular reprogramming allowsthe production of stem cells, replacement cells, tissues, and organsthat match individual patients. In conjunction with gene therapytechniques and cell culture techniques, cell type interconversion alsoprovides for the production of disease-resistant andgenetically-repaired cells that are suitable for transplantation.

It is an object of this invention to provide various manners ofgenerating proliferating, self-renewing, totipotent, multipotent and/orpluripotent cell population(s), as well as other desirabledifferentiating/differentiated cell populations, from either dividing ornon-dividing cells without the use of oncogenes. Differentiating cellpopulations comprise cells expressing some, but not all markersassociated with specific cell type categorization. It is disclosedherein, for example, that appropriate Numb isoform expression incombination with other transgenes/proteins (especially transcriptionfactors) enables the production of dividing, pluripotent cellpopulations or differentiating cell populations. Moreover, the geneticvectors of the present invention may be used to produce geneticmodification (e.g. expression of gene products deficient in the patient)and to transiently or permanently induce proliferation, self-renewal, orstem/progenitor cell behavior in endogenous cells in vivo, particularlythose cells found in tissues which normally do not show or no longershow such behavior. Finally, other genetic vectors of the presentinvention may be used to produce genetic modification and/or to blockproliferation, self-renewal, or stem/progenitor cell behavior in cellsaberrantly displaying such behavior (e.g. cancer cells). It is also anobject of the present invention to provide therapeutic vectors and cellscapable of expressing synthetic oligonucleotide sequences predicted toattenuate disease processes. For example, the current inventiondiscloses the use of synthetic oligonucleotides to reduce geneexpression critical HIV and other immunodeficiency virus infection,propagation and spread.

The invention may be used with any suitable cells, including vertebratecells, and including fish, mammalian, avian, amphibian, and reptiliancells.

The inventor observed 100% efficiency in cell reprogramming with proteinelectroporation-orders of magnitude above other reported methods.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a front view of a two well cell or tissue culture dishconstructed according to this invention, containing different cell ortissue cultures within each separate well.

FIG. 2 is a front view of a two well cell or tissue culture dishconstructed according to this invention with the wells positionedside-by-side.

FIG. 3 is a front view of a three well cell or tissue culture dishconstructed according to this invention with the wells positionedside-by-side.

FIG. 4 is a front view of a nine well cell or tissue culture dishconstructed according to this invention with the wells positionedside-by-side.

FIG. 5 is a schematized vector map corresponding to the vector sequenceof Example 13.

DETAILED DESCRIPTION OF FIGURES

The multi-chambered cell or tissue culture dish shown in FIG. 1 iscomposed of a common base 1 made of the same transparent chemicalresistant material. In the present embodiment, two wells are formed asdepicted, the center well being defined by well wall 3 and the surroundwell being defined by well wall 2 which is dimensionally higher, as canbe determined by measuring from the base 1 to the surround cell wall rim4 than is well wall 3 which is measured from the base 1 to the centercell wall rim 5. It is understood that a greater number of center wellsmay be provided depending on the application having differing cell wallheights. FIG. 1 also depicts a cell or tissue culture 7, situated in thesurround well, which is defined dimensionally by the base 1, thesurround well wall 2 and the center well wall 3. FIG. 1 also depicts acell or tissue culture 6, situated in the center well as defineddimensionally by the base 1, and the center well wall 3.

The multi-chambered cell or tissue culture dish shown in FIG. 2 iscomposed of a common base 5 made of the same transparent chemicalresistant material. In the present embodiment, two wells are formed asdepicted, the left well being separated from the right well by well wall1 and the right well being defined by well walls 1, 2, 3, and 4. It isunderstood that a greater number of wells than the two depicted may bejuxtaposed together depending on the application. FIG. 2 also depicts acell or tissue culture 6 situated in the left well.

The multi-chambered cell or tissue culture dish shown in FIG. 3 iscomposed of a common base 6 made of the same transparent chemicalresistant material. In the present embodiment, three wells are formed asdepicted, the right well being separated from the middle well by wellwall 2 and the right well being defined by well walls 2, 3, 4, and 5. Itis understood that a greater number of wells than the three depicted maybe juxtaposed together depending on the application. FIG. 3 also depictsa cell or tissue culture 7 situated in the far left well.

The multi-chambered cell or tissue culture dish shown in FIG. 4 iscomposed of a common base 15 made of the same transparent chemicalresistant material as the remainder of well walls. In the presentembodiment, nine wells are formed as depicted, the right well beingseparated from the middle well by well wall 2 and the first well beingdefined by well walls 24, 3, 4, and 1. the second well being defined bywell walls 4, 5, 6, and 7, the third well being defined by well walls 7,8, 9, and 10, the fourth well being defined by well walls 6, 8, 12, and14, the fifth well being defined by well walls 14, 14, 16, and a frontwall, the sixth well being defined by well walls 16, 17, 18 and a rearwall, the seventh well being defined by well walls 18, 19, 20 and 21,the eighth well being defined by well walls 21, 22, 23 and an interiorwell wall, and the ninth well being defined as the well situated in thecenter and surrounded by wells 1 through 8, sharing common walls withthose wells. It is understood that a greater number of wells than thenine depicted may be juxtaposed together depending on the application.FIG. 4 also depicts a cell or tissue culture 13 situated in the fourthwell.

It will be clear to a person skilled in the art that specificembodiments discussed herein are not the only possible modes of thisinvention that can be manufactured. Many other features that are notshown in the described embodiments are within the scope of thisinvention.

DETAILED DESCRIPTION

All patents, patent applications, and publications cited in thisapplication are hereby incorporated by reference herein in theirentireties.

As discussed herein, ‘“DNA” refers to deoxyribonucleic acid and “RNA”refers to ribonucleic acid. As discussed herein, “cDNA” refers tocomplementary DNA; “mRNA” refers to messenger RNA; “siRNA” refers tosmall interfering RNA; “shRNA” refers to small hairpin RNA; “miRNA’”refers to microRNA, such as single-stranded RNA molecules, typicallyabout 20-30 nucleotides in length, which may regulate gene expression;“decoy” and “decoy RNA” and “RNA decoy” refer to an RNA molecule thatmimics the natural binding domain for a ligand.

As used herein, the meaning of the term “ameliorating” includeslessening an effect, or reducing damage, or minimizing the effect orimpact of an action, activity, or function, and includes, for example,lessening the deleterious effects of a disease or condition.

As used herein, the meaning of the term “retarding” includes slowing orlessening the progress of an effect or action, and includes, forexample, slowing the progress of a disease, slowing the rate ofinfection, or otherwise acting to slow or reduce the advance or progressof a disease or condition.

As used herein, an “inducing agent” is an agent that aids or is aloneeffective to promote an action. For example, an exogenous agent thataffects a promoter, e.g., by initiating or enhancing its activity, andso affects expression of a gene under control of the promoter, may betermed an inducing agent. For example, tetracycline may be used as aninducing agent; and doxycycline may be used as an inducing agent.

A nucleic acid sequence (e.g., a nucleic acid sequence encoding apolypeptide) is termed “operably linked” to another nucleic acidsequence (e.g., a promoter) when the first nucleic acid sequence isplaced in a functional relationship with the second nuceleic acidsequence. For example, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. As used herein, the term “driven by” refers to a geneor coding sequence that is operably linked to a promoter sequence, andthat the promoter sequence affects the transcription or expression ofthe coding sequence.

As used herein, a “marker” is a molecule that is detectable, or codesfor a detectable molecule, or acts on other molecules so that thepresence of the marker is detectable. A “marker protein” or “markerpolypeptide” is a protein or polypeptide that is detectable in alaboratory or clinical environment, and, in embodiments, may bedetectable by eye. A “marker gene” encodes a marker protein or markerpolypeptide.

As used herein, “HIV” refers to human immunodeficiency virus, andincludes variants such as, e.g., HIV-I, HIV-2. Other immunodeficiencyviruses include simian immunodeficiency virus (SIV) and felineimmunodeficiency virus (FIV). Enzymes related to IHV may be termed “HIVenzymes” and include, for example,\integrase, protease, reversetranscriptase, and transactivating regulatory protein (TAT).

Infection by HIV is believed to involve receptors termed “HIVreceptors.” There may be multiple such receptors, some of which may betermed “HIV co-receptors.” As discussed herein, HIV co-receptors includeCXCR4 and CCR5.

A theoretical basis for the embodiments of the invention is describedherein, however, this discussion is not in any way to be considered asbinding or limiting on the present invention. Those of skill in the artwill understand that the various embodiments of the invention may bepracticed regardless of the model used to describe the theoreticalunderpinnings of the invention.

In a preferred embodiment, cells are “selected” from accessible,dividing or non-dividing cell populations for the purpose of generatingthe desired a) proliferating, multipotent or pluripotent cellpopulation, differentiating b) populations of neuronal cells c) musclecells, d) and/or any other desired cell population; moreover the desiredcell population may be capable of further differentiation in vitro, invivo, and/or tissue-appropriate and regionally-appropriatedifferentiation in vivo.

Sources of cells selected for use in the invention:

Selected cells may include any cell practicable in the presentinvention. Cells selected for use in the present invention (hereintermed “selected cells”) may originate as endogenous cells of thepatient—including cells derived from other organ systems; or fromexogenous sources (including those derived from cell lines,cryopreserved sources, banked sources, and donors). Cells may also beselected from cells genetically-modified with synthetic or naturalnucleic acid sequences. The term “selected cells” as used herein doesnot include human embryonic stem cells.

In embodiments of the present invention, in order that they may beisolated without the involvement of invasive procedures, selected cellswill preferably be easily accessible cells (e.g. peripheral bloodleukocytes, circulating hematopoietic stem cells, epithelial cells (e.g.buccal cheek cells (e.g. Michalczyk et al, 2004)), adipose tissue (e.g.Gimble et al., 2007; Ma et al., 2007), umbilical cord blood cells (e.g.Zhao, et al., 2006; Tian et al., 2007), etc.). However, bone marrow stemcells, spermatogonia (e.g. Guan el al., 2006; Takahashi et al., 2007),primordial germ cells (PGCs), stem cells isolated from amnioticmembranes (e.g. Ilancheran et al., 2007), amniotic fluid (e.g. De Coppiet al., 2007), as well as cells isolated from the skin (e.g. Tumbar,2006; Dunnwald et al., 2001; Szudal'tseva et al., 2007), etc., are alsocovered by the present invention. Such cells can be isolated from thetissues in which they reside by any means known to the art.

Spermatogonia cells can be isolated using a two-step enzymatic digestionfollowed by Percoll separation. Cells can then be resuspended in minimumessential medium (MEM) supplemented with bovine serum albumin to a finalconcentration of 106/mL. In detail: Tubule fragments are accessedsurgically and teased apart prior to treatment with 1 mg/ml trypsin,hyaluronidase, and collagenase, and then 1 mg/ml hyaluronidase andcollagenase, in MEM containing 0.10% sodium bicarbonate, 4 mML-glutamine, nonessential amino acids, 40 microgram/ml gentamycin, 100KJ to 100 microgram/ml penicillin-streptomycin, and 15 mM HEPES.Spermatogonia cells are further separated from tubule fragments bycentrifugation at 30 times gravity. After filtration through nylonfilters with 77- and/or 55-micron pore sizes, cells are collected andloaded onto a discontinuous Percoll density gradient. Fractions with apurity greater than 40% progenitor/stem/spermatogonia cells are washedand resuspended to a concentration of cells equivalent to 10progenitor/stem/spermatogonia cells per ml. Afterwards cells arecultured and/or stored by any cryopreservation technique known to theart.

The selected cells may be genetically-modified cells, especially cellsthat have been genetically modified by any means known to the art, toencode therapeutic or commercially useful deoxyribonucleic acid (DNA) orribonucleic acid (RNA) sequences.

In accordance with an aspect of the present invention, there is provideda method of producing a desired cell population (e.g. pluripotent,neuronal, muscle, etc.) from the selected cells.

Achieving multipotent, pluripotent, and/or self renewing cellpopulations:

In order to achieve a) a population of proliferating, self renewingpluripotent cells, the selected cell(s) and/or their progeny aretransfected/contacted with nucleotide sequence(s) including thoseencoding the “long” (PRR insert+) isoform(s) of the mammalian numb gene.At about the same time the selected cells may also betransfected/contacted with synthetic oligonucleotides targeting theshort Numb isoforms and Numblike, then cultured under conditions whichpromote growth of the selected cells at an optimal growth rate. Selectedcells are maintained under these conditions for the period of timesufficient to achieve the desired cell number.

The cells are grown at the (optimal) rate of growth achieved byincubation with LIF, steel factor, and/or equipotent concentrations of11-6, hyper 1L-6, IL-7, oncostatin-M and/or cardiotrophin-1; or thatgrowth rate achieved in the presence of other growth enhancing cytokines(e.g. those conditions described for culturing pluripotent cells e.g.Guan et al., 2006). The growth rate is determined from the doublingtimes of the selected cells in said growth culture medium. Likewise,culture conditions such as those described in U.S. Pat. Nos. 6,432,711and 5,453,357 may also be suitable for the propagation and expansion, atan optimal growth rate, of cells transfected/contacted with the long(PRR+) Numb isoform(s). Other appropriate protocols and referencecytokine concentrations have been taught by Koshimizu et al., 1996;Keller et al., 1996; Piquet-Pellorce, 1994; Rose et al., 1994; Park andHan, 2000; Guan et al., 2006; Dykstra et al., 2006; Zhang et al., 2007).However the practice of the present invention is not limited to thedetails of these teachings.

In a preferred embodiment, the selected cells are cultured in a standardgrowth medium (e.g. Minimal Essential Medium with or without supplements(e.g. glutamine, and beta.-mercaptoethanol). The medium may includebasic fibroblast growth factor (bFGF), steel factor, leukemia inhibitoryfactor (LIF), and/or factors with LIF activity (e.g. LIF, LIF receptor(LIFR), ciliary Neurotrophic factor (CNTF), oncostatin M (OSM), OSMreceptor (OSMR), cardiotrophin, interleukins (IL) such as IL-6, hyperIL-6, GP 130, etc.) as well as horse serum. LIF, as well as otherfactors with LIF activity, prevents spontaneous differentiation of thecells. Under these conditions, selected cells transfected/contacted withthe PRR+Numb isoform(s) and their progeny are expected to achievemultipotency, pluripotency and/or self-renewal.

In a preferred embodiment, the selected cell(s) and/or their progeny aretransfected/contacted with nucleotide sequence(s) encoding the “long”(PRR insert+) Numb isoform(s) as well as sequences encoding othertransgenes. Many of those transgenes are listed below along with theircorresponding identification numbers (accession numbers) in the NCBIsequence database.

In another preferred embodiment, the selected cell(s) and/or theirprogeny are transfected with nucleotide sequence(s) encoding a portionof the “long” (PRR insert+) Numb isoform(s) as well as sequencesencoding other transgenes. Many of those transgenes are listed belowalong with their corresponding identification (accession) numbers(codes) in the NCBI sequence database.

In a preferred embodiment, the selected cells and/or their progeny aretransfected/contacted with long (PRR+) Numb isoform encoding sequencesas well as sequences encoding other transgenes, including LIF.

In a preferred embodiment, the selected cells and/or their progeny aretransfected/contacted with long (PRR+) Numb isoform encoding sequencesas well as sequences encoding other transgenes, including ones with LIFactivity.

In a preferred embodiment, the selected cells and/or their progeny aretransfected/contacted with long (PRR+) Numb isoform(s) encodingsequences as well as sequences encoding other transgenes, including theLIFR.

In a preferred embodiment, the selected cells and/or their progeny aretransfected/contacted with long (P RR+) Numb isoform(s) encodingsequences as well as sequences encoding other transgenes, includingoncostatin M (OSM).

In a preferred embodiment, the selected cells and/or their progeny aretransfected/contacted with long (PRR+) Numb isoform(s) encodingsequences as well as sequences encoding other transgenes, includingoncostatin M receptor (OSMR).

In a preferred embodiment, the selected cells and/or their progeny aretransfected/contacted with long (PRR+) Numb isoform(s) encodingsequences as well as sequences encoding other transgenes, includingcardiotrophin-1.

In a preferred embodiment, the selected cells and/or their progeny aretransfected/contacted with long (PRR+) Numb isoform(s) encodingsequences as well as sequences encoding other transgenes, includingCNTF.

In a preferred embodiment, the selected cells and/or their progeny aretransfected/contacted with long (PRR+) Numb isoform(s) encodingsequences as well as sequences encoding other transgenes, includingOCT3/4 and SOX2.

In a preferred embodiment, the selected cells and/or their progeny aretransfected/contacted with long (PRRf) Numb isoform(s) encodingsequences as well as sequences encoding other transgenes, includingNANOG, OCT3/4 and SOX2.

In a preferred embodiment, the selected cells and/or their progeny aretransfected/contacted with long (PRR+) Numb isoform(s) encodingsequences as well as sequences encoding other transgenes, includingOCT3/4 and SOX2 and a transgene with LIF activity.

In a preferred embodiment, the selected cells and/or their progeny aretransfected/contacted sequences encoding other transgenes, includingOCT3/4 and SOX2 and a transgene with LIF activity.

In a preferred embodiment, the selected cells and/or their progeny aretransfected/contacted with long (PRR+) Numb isoform(s) encodingsequences as well as sequences encoding other transgenes, includingNotch (e.g. Gaiano et al., 2000).

In a preferred embodiment, the selected cells and/or their progeny aretransfected/contacted with long (PRR+) Numb isoform(s) encodingsequences as well as sequences encoding other transgenes, includingOCT3/4, SOX2 and Notch (e.g. notch 1 and/or notch 2).

In a preferred embodiment, the selected cells and/or their progeny aretransfected/contacted with long (PRR+) Numb isoform(s) encodingsequences as well as sequences encoding other transgenes, includingOCT3/4, SOX2, NANOG, and Notch.

In a preferred embodiment, the selected cells and/or their progeny aretransfected/contacted with long (P RR+) Numb isoform(s) encodingsequences as well as sequences encoding other transgenes, includingOCT3/4, SOX2, NANOG, and a transgene with LIF activity.

In a preferred embodiment, the selected cells and/or their progeny aretransfected/contacted with long (PRR+) Numb isoform(s) encodingsequences as well as sequences encoding other transgenes, includingOCT3/4, SOX2, NANOG, and multiple transgenes with LIF activity.

In a preferred embodiment, the selected cells and/or their progeny aretransfected/contacted with long (PRR+) Numb isoform(s) encodingsequences as well as sequences encoding other transgenes, includingOCT3/4, Notch, HOXB4 and SOX2.

Over time, other gene combinations differing from those described hereinmay be described or discovered capable of causing cells to becomemultipotent, pluripotent, capable of self-renewal, or to begindifferentiating. However this patent application covers such “‘geneticreprogramming” of any nucleated cell utilizing nucleic acid or proteinelectroporation (see Gagne et al., 1991; Saito et al., 2001; Yuan, 2008;Huang et al., 2007; Xia and Zhang, 2007; Cemazar and Sersa 2007; lsakaand Imai, 2007; Luxembourg et al., 2007; Van Tendeloos, 2007; Takahashi,2007; etc.), liposomes, nanocapsules, nanovaults, etc. (see Goldberg etal., 2007; Li et al., 2007), and/or another approach avoiding viralintegration or other random alteration of the cell's genome, as suchmeans increase safety and efficiency.

Excluded, of course, from the category of random alteration areapproaches involving gene-targeting and site-directed methods designedto introduce or remove DNA at specific locations in the genome.

Likewise, this patent application covers the genetic reprogramming ofany nucleated cell utilizing nucleic acid or protein electroporation,liposomes, nanocapsules, nanovaults, etc., and/or another approachavoiding retroviral/lenti viral integration or other random alterationof the cell's genome, as such means increase safety and efficiency. Suchapproaches and methods include all known to the art and practicable inthe present invention.

In a preferred embodiment, nucleic acid(s) or protein(s) correspondingto a single gene, or portion thereof, (particularly those named herein,discovered according to methods described herein, discovered accordingto other published methods; or known to be multipotency, pluripotency,or self-renewal inducing) are the only nucleic acid(s) or protein(s)overexpressed and/or introduced to produce multipotent, pluripotent,and/or self-renewing cells from the selected cells.

In a preferred embodiment, nucleic acid(s) or protein(s) correspondingto a single gene, or portion thereof, (particularly those named herein,discovered according to methods described herein, discovered accordingto other published methods; or known to be multipotency, pluripotency,or self-renewal inducing) are the only nucleic acid(s) or protein(s)overexpressed and/or introduced to produce multipotent, pluripotent,and/or self-renewing cells from the selected cells and the methodutilized is electroporation, liposomes, nanocapsules, nanovaults, and/oranother approach avoiding retroviral/lentiviral integration or otherrandom alteration of the cell's genome.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)can be utilized in concert with the nucleic acid(s) or protein(s)corresponding to a single gene, or portion thereof, (particularly thosenamed herein, discovered according to methods described herein,discovered according to other published methods; or known to bemultipotency, pluripotency, or self-renewal inducing) so long as apopulation of multipotent, pluripotent, and/or self-renewing cells isproduced from the selected cells.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)can be utilized in concert with the nucleic acid(s) or protein(s)corresponding to a single gene, or portion thereof, (particularly thosenamed herein, discovered according to methods described herein,discovered according to other published methods; or known to bemultipotency, pluripotency, or self-renewal inducing) so long as apopulation of multipotent, pluripotent, and/or self-renewing cells isproduced from the selected cells and the method utilized iselectroporation, liposomes, nanocapsules, nanovaults, and/or anotherapproach avoiding retroviral/lentiviral integration or other randomalteration of the cell's genome.

In a preferred embodiment, nucleic acid(s) or protein(s) correspondingto Nanog are the only nucleic acid(s) or protein(s) overexpressed and/orintroduced to produce multipotent, pluripotent, and/or self-renewingcells from the selected cells and the method utilized iselectroporation, liposomes, nanocapsules, nanovaults, and/or anotherapproach avoiding retroviral/lentiviral integration or other randomalteration of the cell's genome.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)can be utilized in concert with the nucleic acid(s) or protein(s)corresponding to Nanog so long as a population of multipotent,pluripotent, and/or self-renewing cells is produced from the selectedcells and the method utilized is electroporation, liposomes,nanocapsules, nanovaults, and/or another approach avoiding viralintegration or other random alteration of the cell's genome.

In a separate preferred embodiment, nucleic acid(s) or protein(s)corresponding to Oct4 and Sox2 are the only nucleic acid(s) orprotein(s) overexpressed and/or introduced to produce multipotent,pluripotent, and/or self-renewing cells from the selected cells and themethod utilized is electroporation, liposomes, nanocapsules, nanovaults,and/or another approach avoiding viral integration or other randomalteration of the cell's genome.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)are utilized in concert with the nucleic acid(s) or protein(s)corresponding to Oct4/Sox2 so long as a population of multipotent,pluripotent, and/or self-renewing cells is produced from the selectedcells and the method utilized is electroporation, liposomes,nanocapsules, nanovaults, and/or another approach avoiding viralintegration or other random alteration of the cell's genome.

In a separate preferred embodiment, nucleic acid(s) or protein(s)corresponding to Long (PRR+) Numb isoforms are the only nucleic acid(s)or protein(s) overexpressed and/or introduced to produce multipotent,pluripotent, and/or self-renewing cells from the selected cells and themethod utilized is electroporation, liposomes, nanocapsules, nanovaults,and/or another approach avoiding viral integration or other randomalteration of the cell's genome.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)are utilized in concert with the nucleic acid(s) or protein(s)corresponding to Long (PRR+) Numb isoforms so long as a population ofmultipotent, pluripotent, and/or self-renewing cells is produced fromthe selected cells and the method is electroporation, liposomes,nanocapsules, nanovaults, and/or another approach avoiding viralintegration or other random alteration of the cell's genome.

In a preferred embodiment, nucleic acid(s) or protein(s) correspondingto Nanog are the only nucleic acid(s) or protein(s) overexpressed and/orintroduced to produce multipotent, pluripotent, and/or self-renewingcells from the selected cells.

In a preferred embodiment, nucleic acid(s) or protein(s) correspondingto Nanog are the only nucleic acid(s) or protein(s) overexpressed and/orintroduced to produce multipotent, pluripotent, and/or self-renewingcells from the selected cells and the method utilized iselectroporation, liposomes, nanocapsules, nanovaults, and/or anotherapproach avoiding retroviral/lentiviral integration or other randomalteration of the cell's genome.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)can be utilized in concert with the nucleic acid(s) or protein(s)corresponding to Nanog so long as a population of multipotent,pluripotent, and/or self-renewing cells is produced from the selectedcells.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)can be utilized in concert with the nucleic acid(s) or protein(s)corresponding to Nanog so long as a population of multipotent,pluripotent, and/or self-renewing cells is produced from the selectedcells and the method utilized is electroporation, liposomes,nanocapsules, nanovaults, and/or another approach avoidingretroviral/lentiviral integration or other random alteration of thecell's genome.

In a preferred embodiment, nucleic acid(s) or protein(s) correspondingto a gene with LIF activity are the only nucleic acid(s) or protein(s)overexpressed and/or introduced to produce multipotent, pluripotent,and/or self-renewing cells from the selected cells.

In a preferred embodiment, nucleic acid(s) or protein(s) correspondingto a gene with LIF activity are the only nucleic acid(s) or protein(s)overexpressed and/or introduced to produce multipotent, pluripotent,and/or self-renewing cells from the selected cells and the methodutilized is electroporation, liposomes, nanocapsules, nanovaults, and/oranother approach avoiding retroviral/lentiviral integration or otherrandom alteration of the cell's genome.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)can be utilized in concert with the nucleic acid(s) or protein(s)corresponding to to a gene with LIF activity so long as a population ofmultipotent, pluripotent, and/or self-renewing cells is produced fromthe selected cells.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)can be utilized in concert with the nucleic acid(s) or protein(s)corresponding to a gene with LIF activity so long as a population ofmultipotent, pluripotent, and/or self-renewing cells is produced fromthe selected cells and the method utilized is electroporation,liposomes, nanocapsules, nanovaults, and/or another approach avoidingretroviral/lentiviral integration or other random alteration of thecell's genome.

In a preferred embodiment, nucleic acid(s) or protein(s) correspondingto Oct4 are the only nucleic acid(s) or protein(s) overexpressed and/orintroduced to produce multipotent, pluripotent, and/or self-renewingcells from the selected cells.

In a preferred embodiment, nucleic acid(s) or protein(s) correspondingto Oct4 are the only nucleic acid(s) or protein(s) overexpressed and/orintroduced to produce multipotent, pluripotent, and/or self-renewingcells from the selected cells and the method utilized iselectroporation, liposomes, nanocapsules, nanovaults, and/or anotherapproach avoiding re troviral/lenti viral integration or other randomalteration of the cell's genome.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)can be utilized in concert with the nucleic acid(s) or protein(s)corresponding to Oct4 so long as a population of multipotent,pluripotent, and/or self-renewing cells is produced from the selectedcells.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)can be utilized in concert with the nucleic acid(s) or protein(s)corresponding to Oct4 so long as a population of multipotent,pluripotent, and/or self-renewing cells is produced from the selectedcells and the method utilized is electroporation, liposomes,nanocapsules, nanovaults, and/or another approach avoidingretroviral/lenti viral integration or other random alteration of thecell's genome.

In a preferred embodiment, nucleic acid(s) or protein(s) correspondingto Sox2 are the only nucleic acid(s) or protein(s) overexpressed and/orintroduced to produce multipotent, pluripotent, and/or self-renewingcells from the selected cells.

In a preferred embodiment, nucleic acid(s) or protein(s) correspondingto Sox2 are the only nucleic acid(s) or protein(s) overexpressed and/orintroduced to produce multipotent, pluripotent, and/or self-renewingcells from the selected cells and the method utilized iselectroporation, liposomes, nanocapsules, nanovaults, and/or anotherapproach avoiding retroviral/lentiviral integration or other randomalteration of the cell's genome.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)can be utilized in concert with the nucleic acid(s) or protein(s)corresponding to Sox2 so long as a population of multipotent,pluripotent, and/or self-renewing cells is produced from the selectedcells.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)can be utilized in concert with the nucleic acid(s) or protein(s)corresponding to Sox2 so long as a population of multipotent,pluripotent, and/or self-renewing cells is produced from the selectedcells and the method utilized is electroporation, liposomes,nanocapsules, nanovaults, and/or another approach avoidingretroviral/lentiviral integration or other random alteration of thecell's genome.

In a preferred embodiment, nucleic acid(s) or protein(s) correspondingto Iin28 are the only nucleic acid(s) or protein(s) overexpressed and/orintroduced to produce multipotent, pluripotent, and/or self-renewingcells from the selected cells.

In a preferred embodiment, nucleic acid(s) or protein(s) correspondingto Iin28 are the only nucleic acid(s) or protein(s) overexpressed and/orintroduced to produce multipotent, pluripotent, and/or self-renewingcells from the selected cells and the method utilized iselectroporation, liposomes, nanocapsules, nanovaults. and/or anotherapproach avoiding retroviral/lentiviral integration or other randomalteration of the cell's genome.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)can be utilized in concert with the nucleic acid(s) or protein(s)corresponding to Iin28 so long as a population of multipotent,pluripotent, and/or self-renewing cells is produced from the selectedcells and the method utilized is electroporation, liposomes,nanocapsules, nanovaults, and/or another approach avoidingretroviral/lentiviral integration or other random alteration of thecell's genome.

In a preferred embodiment, nucleic acid(s) or protein(s) correspondingto c-myc are the only nucleic acid(s) or protein(s) overexpressed and/orintroduced to produce multipotent, pluripotent, and/or self-renewingcells from the selected cells.

In a preferred embodiment, nucleic acid(s) or protein(s) correspondingto c-myc are the only nucleic acid(s) or protein(s) overexpressed and/orintroduced to produce multipotent, pluripotent, and/or self-renewingcells from the selected cells and the method utilized iselectroporation, liposomes, nanocapsules, nanovaults, and/or anotherapproach avoiding retroviral/lentiviral integration or other randomalteration of the cell's genome.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)can be utilized in concert with the nucleic acid(s) or protein(s)corresponding to c-myc so long as a population of multipotent,pluripotent, and/or self-renewing cells is produced from the selectedcells.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)can be utilized in concert with the nucleic acid(s) or protein(s)corresponding to c-myc so long as a population of multipotent,pluripotent, and/or self-renewing cells is produced from the selectedcells and the method utilized is electroporation, liposomes,nanocapsules, nanovaults, and/or another approach avoidingretroviral/lentiviral integration or other random alteration of thecell's genome.

In a separate preferred embodiment, nucleic acid(s) or protein(s)corresponding Oct4 and Sox2 are the only nucleic acid(s) or protein(s)overexpressed and/or introduced to produce multipotent, pluripotent,and/or self-renewing cells from the selected cells.

In a separate preferred embodiment, nucleic acid(s) or protein(s)corresponding to Oct4 and Sox2 are the only nucleic acid(s) orprotein(s) overexpressed and/or introduced to produce multipotent,pluripotent, and/or self-renewing cells from the selected cells and themethod utilized is electroporation, liposomes, nanocapsules, nanovaults,and/or another approach avoiding retroviral/lentiviral integration orother random alteration of the cell's genome.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)are utilized in concert with the nucleic acid(s) or protein(s)corresponding to Oct4 and Sox2 so long as a population of multipotent,pluripotent, and/or self-renewing cells is produced from the selectedcells.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)are utilized in concert with the nucleic acid(s) or protein(s)corresponding to Oct4 and Sox2 so long as a population of multipotent,pluripotent, and/or self-renewing cells is produced from the selectedcells and the method utilized is electroporation, liposomes,nanocapsules, nanovaults, and/or another approach avoidingretroviral/lentiviral integration or other random alteration of thecell's genome.

In a separate preferred embodiment, nucleic acid(s) or protein(s)corresponding to Long (PRR+) Numb isoforms are the only nucleic acid(s)or protein(s) overexpressed and/or introduced to produce multipotent,pluripotent, and/or self-renewing cells from the selected cells.

In a separate preferred embodiment, nucleic acid(s) or protein(s)corresponding to Long (PRR+) Numb isoforms are the only nucleic acid(s)or protein(s) overexpressed and/or introduced to produce multipotent,pluripotent, and/or self-renewing cells from the selected cells and themethod utilized is electroporation, liposomes, nanocapsules, nanovaults,and/or another approach avoiding retroviral/lentiviral integration orother random alteration of the cell's genome.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)are utilized in concert with the nucleic acid(s) or protein(s)corresponding to Long (PRR+) Numb Isoforms so long as a population ofmultipotent, pluripotent, and/or self-renewing cells is produced fromthe selected cells.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)are utilized in concert with the nucleic acid(s) or protein(s)corresponding to Long (PRR+) Numb Isoforms so long as a population ofmultipotent, pluripotent, and/or self-renewing cells is produced fromthe selected cells and the method utilized is electroporation,liposomes, nanocapsules, nanovaults, and/or another approach avoidingretroviral/lentiviral integration or other random alteration of thecell's genome.

In a separate preferred embodiment, nucleic acid(s) or protein(s)corresponding to Oct4, Sox2, and Nanog are the only nucleic acid(s) orprotein(s) overexpressed and/or introduced to produce multipotent,pluripotent, and/or self-renewing cells from the selected cells.

In a separate preferred embodiment, nucleic acid(s) or protein(s)corresponding to Oct4, Sox2, and Nanog are the only nucleic acid(s) orprotein(s) overexpressed and/or introduced to produce multipotent,pluripotent, and/or self-renewing cells from the selected cells and themethod utilized is electroporation, liposomes, nanocapsules, nanovaults,and/or another approach avoiding retroviral/lentiviral integration orother random alteration of the cell's genome.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)are utilized in concert with the nucleic acid(s) or protein(s)corresponding to Oct4, Sox2, and Nanog so long as a population ofmultipotent, pluripotent, and/or self-renewing cells is produced fromthe selected cells.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)are utilized in concert with the nucleic acid(s) or protein(s)corresponding to Oct4, Sox2, and Nanog so long as a population ofmultipotent, pluripotent, and/or self-renewing cells is produced fromthe selected cells and the method utilized is electroporation,liposomes, nanocapsules, nanovaults, and/or another approach avoidingretroviral/lentiviral integration or other random alteration of thecell's genome.

In a separate preferred embodiment, nucleic acid(s) or protein(s)corresponding to Long (PRR+) Numb isoforms are the only nucleic acid(s)or protein(s) overexpressed and/or introduced to produce multipotent,pluripotent. and/or self-renewing cells from the selected cells.

In a separate preferred embodiment, nucleic acid(s) or protein(s)corresponding to Long (PRR+) Numb isoforms are the only nucleic acid(s)or protein(s) overexpressed and/or introduced to produce multipotent,pluripotent, and/or self-renewing cells from the selected cells and themethod is electroporation, liposomes, nanocapsules, nanovaults, and/oranother approach avoiding retroviral/lentiviral integration or otherrandom alteration of the cell's genome.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)are utilized in concert with the nucleic acid(s) or protein(s)corresponding to Long (PRR+) Numb isoforms so long as a population ofmultipotent, pluripotent, and/or self-renewing cells is produced fromthe selected cells.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)are utilized in concert with the nucleic acid(s) or protein(s)corresponding to Long (PRR+) Numb isoforms so long as a population ofmultipotent, pluripotent, and/or self-renewing cells is produced fromthe selected cells and the method is electroporation, liposomes,nanocapsules, nanovaults, and/or another approach avoidingretroviral/lentiviral integration or other random alteration of thecell's genome.

It is to be understood that any combination of nucleic acid or proteinsequences described herein can be modified by excluding thosecorresponding to Numb and/or Numblike so long as the desired cellpopulation or behavior is achieved.

Similarly, it should be understood that the methods described herein forinitiating differentiation are applicable to any induced or non-inducedmultipotent, pluripotent, or self-renewing stem cells, other progenitorcells, or other selected cells, not only those obtained in the mannerdescribed herein.

It is to be understood that any combination of nucleic acid or proteinsequences described herein can be modified by excluding nucleic acidsequences or proteins corresponding to Numb and/or Numblike so long asthe desired cell population is achieved.

In another embodiment, the various nucleic acid or protein combinationsdescribed herein are employed with the exclusion of the nucleic acid orprotein corresponding to the Numblike and/or Numb isoforms.

In a preferred embodiment, the selected cells and/or their progeny arecells that have been genetically-modified beforehand.

In a preferred embodiment, the transfection/contacting steps describedherein represent transient transfection.

In a further preferred embodiment such transient transfection isaccomplished using viral vectors that do not integrate into the hostgenome.

In another preferred embodiment, such transient transfection isaccomplished using standard transfection techniques (electroporation,chemically mediated transfection, fusogenic or non-fusogenic liposomes,nanocapsules, nanovaults, etc.).

Over time, other gene combinations differing from those described hereinmay be described or discovered capable of causing cells to becomemultipotent, pluripotent, capable of self-renewal or to begindifferentiating. However this patent application also covers the geneticreprogramming of any nucleated cell utilizing nucleic acid or proteinelectroporation (for example methods see Gagne et al., 1991; Saito etal., 2001; Yuan, 2008; Huang et al., 2007; Xia and Zhang, 2007; Cemazarand Sersa 2007; Isaka and Imai, 2007; Luxembourg et al., 2007; VanTendeloos, 2007; Takahashi, 2007; etc.) electroporation, liposomes,nanocapsules, nanovaults, and/or another approach avoiding viralintegration or other random alteration of the cell's genome as suchmeans increase safety and efficiency.

In another preferred embodiment, transfection/contacting with long(PRR+) numb isoform encoding sequences (and/or syntheticoligonucleotides targeting numblike and short numb isoforms) isaccompanied or replaced by transient or permanent transfection withother sequences including ones selected from those encoding human LIF(e.g. Du and Shi, 1996) oncostatin-M, cardiotrophin-1, IL-I 1, IL-6,IL6R, hyper IL-6, LIFR, gp130, OCT3 (OCT4), Nanog, SOX2, and/or FGF-4.

Simultaneous transfection/contacting with any subset of these distincttransgene sequences can be accomplished by any means known to the artincluding the use of a single genetic vector, multiple genetic vectors,serial transfection/contacting and selection based on distinct markerproteins and/or antibiotic resistances.

In another preferred embodiment, cells transfected/contacted with long(PRR+) numb isoform(s) are cultured in a cell culture promoting anoptimal growth rate, such as described above, and that includes EGF,bFGF, oncostatin, LIF (e.g. Du and Shi, 1996), steel factor, IL-11,cardiotrophin-1, IL-6, hyper-IL-6, CNTF, and/or soluble gpl30.

Assessment of Potency and Differentiation

Pluripotency and multipotency can be assessed by any means known to theart including 1) transplantation, 2) culture under conditions promotingembryoid body formation, 3) injection of cells into animal blastocyststage embryos with subsequent development, and 4) RNA expression assays(e.g. RI-PCR and microarray based analyses) for gene expressionassociated with differentiation, multipotency, pluripotency, etc. (seeGuan et al., 2006), 5) colony-formation, as well as by ES-likemorphology. One approach disclosed herein for detecting pluripotency inselected cells and/or their progeny involves transfection/contactingwith a reporter construct comprising the Nanog promoter operably linkedto a fluorescent protein gene. This allows identification and enrichmentof Nanog expressing cells using Fluorescence Activated Cell Sorting(FACS), etc.

In a preferred embodiment, endogenous cells (e.g. cells surrounding aburn or injury site) are transfected/contacted in vivo with geneticvectors encoding the long (PRR+) numb isoform(s) alone or in conjuctionwith other transgenes named herein to transiently promote renewed orincreased cell proliferation. This approach can also be utilizedclinically in the setting of hypoplastic tissues, disorders wherestem/progenitor cells are abnormally depleted, and other disorders wherethe approach can be shown to be beneficial.

Achieving Differentiating Cell Populations

In order to achieve b) neural c) muscle d) and other cell populationscapable of further environmentally-regulated differentiation in vivo,selected cell(s) and/or their progeny are optionallytransfected/contacted with long (PRR+) Numb isoform sequence(s) and/orsynthetic oligonucleotide sequences and expanded by growth forsufficient time to achieve the desirable number of cell progeny in vitro(as described above).

Following this optional step, the selected cells and/or their progenyare washed free of the cytokines and agents comprising theexpansion/optimal growth media, and are optionally transfected/contactedwith the nucleotide sequence(s) encoding the Numblike gene and/or“short” ‘(PRR−) Numb isoform(s) and/or synthetic oligonucleotidestargeting the long (PRR+) isoforms, etc. (e.g. Zaehres et al., 2005),then cultured under conditions which promote differentiation of theselected cells into the desired cell type(s).

In most instances, the cells are then cultured in the presence of 5-10%fetal bovine serum and agents(s) promoting differentiation of theselected cells and/or their progeny into a desired cell population. Thepresence of the fetal bovine and of the agents(s) provides for growth orproliferation at a rate that is less than the optimal (or expansion)growth rate, and favors differentiation of the cells into a desired cellpopulation. The agents and precise culture conditions are selectedaccording to the desired cell population as described below.

Achieving Neuronal or Neural Cell Populations

When the desired cell population is a neural cell population, thesuccessfully transfected cells are cultured under conditions thatpromote growth at a rate which is less than the optimal rate and in thepresence of agent(s) promoting differentiation of the cells into neuralcells. Conditions promoting differentiation into neurons have beendescribed in numerous publications including (Benninger et al., 2003;Chung et al. 2005; Harkany et al., 2004; Ikeda et al., 2004; Ikeda etal., 2005; Wernig et al., 2002; and Wernig et al., 2004). Furthermore,combining retinoic acid exposure with the presence of additionalcytokines favors specific neuronal cell type differentiation in vitro(e.g. Soundararajan et al., 2006; Soundararajan et al., 2007; U.S. Pat.No. 6,432,711).

In a preferred embodiment, in vitro differentiation of neurons or neuralcells occurs in the presence of 50 ng/mL nerve growth factor (NGF).

In a preferred embodiment, when a neuronal population is the desiredcell population, transfection/contacting with short numb isoform (and/ornumblike) proteins or with sequences encoding short numb isoformproteins (and/or numblike), is accompanied or replaced by transient orpermanent transfection/contacting with other proteins and/or nucleicacid sequences, including ones selected from those encoding Nurrl, REN,Neurogeninl, Neurogenin2, Neurogenic, Mash 1, Phox2b, Phox2a, dlland,Gata3, Shh, FGF8, Lmxlb, Nkx2.2, Petl, Lbxl, and/or Rnx.

In another preferred embodiment, when dopaminergic neurons are thedesired neuronal population, transfection/contacting with short numbisoform (and/or numblike) proteins or with sequences encoding short numbisoform proteins (and/or numblike), is accompanied or replaced bytransient or permanent transfection/contacting with other proteinsand/or nucleic acid sequences, including ones selected from thoseencoding Mashl, Ngn2, Nurrl, Lmxlb, and/or Ptx-3.

In another preferred embodiment, when serotonergic neurons are thedesired neuronal population, transfection/contacting with short numbisoform (and/or numblike) proteins or with sequences encoding short numbisoform proteins (and/or numblike), is accompanied or replaced bytransient or permanent transfection/contacting with other proteinsand/or nucleic acid sequences, including ones selected from thoseencoding Mashl, Phox2b, Lmxlb, Nkx2.2, Gata2, Gata3 and/or Petl.

In another preferred embodiment, when cholinergic neurons are thedesired neuronal population, transfection/contacting with short numbisoform (and/or numblike) proteins or with sequences encoding short numbisoform proteins (and/or numblike), is accompanied or replaced bytransient or permanent transfection/contacting with other proteinsand/or nucleic acid sequences, including ones selected from thoseencoding MASHlIl, Phox2a and/or RKST4.

In another preferred embodiment, when GABAergic neurons are the desiredneuronal population, transfection/contacting with short numb isoform(and/or numblike) proteins or with sequences encoding short numb isoformproteins (and/or numblike), is accompanied or replaced by transient orpermanent transfection/contacting with other proteins and/or nucleicacid sequences, including ones selected from those encoding MASHl,Phox2a and/or REST4, followed, optionally, by culture in mediasupplemented with LIF, Neurotrophin 3 (NT3), and/or nerve growth factor(NGF).

In another preferred embodiment, when noradrenergic neurons are thedesired neuronal population, transfection/contacting with short numbisoform (and/or numblike) proteins or with sequences encoding short numbisoform proteins (and/or numblike), is accompanied or replaced bytransient or permanent transfection/contacting with other proteinsand/or nucleic acid sequences, including ones selected from thoseencoding Mashl, dlland, Phox2a, Phox2b, Gata2 and/or Gata3.

In another preferred embodiment, when GABAergic neurons are the desiredneuronal population, transfection/contacting with short numb isoform(and/or numblike) proteins or with sequences encoding short numb isoformproteins (and/or numblike), is accompanied or replaced by transient orpermanent transfection/contacting with other proteins and/or nucleicacid sequences, including ones selected from those encoding P1TX2, Dlx2,Dlx5, antisense Hesl RNA and/or other HESl targeting syntheticoligonucleotides.

In another preferred embodiment, when a neuronal or neural cellpopulation is the desired population, cells transfected/contacted withshort (PRR−) numb isoforms (and/or numblike) are cultured in a cellculture medium promoting differentiation, such as described above andthat includes one or more of the following agents: retinoic acid, NT3,NGF, glial cell-line derived growth factor (GDNF), and interferon gamma(IFN-gamma)

Achieving Muscle Cell Populations

When the desired cell population is a muscle population, thesuccessfully transfected/contacted cells are cultured in the presence ofan agent promoting differentiation of the cells into muscle cells andgrowth at a rate less than the optimal rate. Conditions promotingdifferentiation into muscle cells have also been described previously(Nakamura et al., 2003; Pal and Khanna, 2005; Pipes et al., 2005;Albilez et al., 2006; Pal and Khanna, 2007; Behfar et al., 2007; U.S.Pat. No. 6,432,711). Furthermore, exposure of selected cells and/ortheir progeny to hexamethylene bis-acrylamide or dimethylsulfoxide inthe presence of additional cytokines favors the initiation of muscletype differentiation in vitro.

In a preferred embodiment, when a cardiac muscle cell population is thedesired population, cells transfected/contacted with short (PRR−) numbisoforms (and/or numblike) are cultured in a cell culture mediumpromoting differentiation into cardiomyocytes (He et al., 2003; Guan etal., 2007; etc.), or that includes specific agents at concentrationspromoting cardiac cell differentiation (e.g. 0.75%-1% dimethyl sulfoxide(DMSO), 20% normal bovine serum (NBS), 10(-7) mM retinoic acid (RA) and20% cardiomyocytes conditioned medium (Hua et al., 2006).

In a preferred embodiment, when a muscle cell population is the desiredcell population, transfection/contacting with short numb isoform (and/ornumblike) proteins or with sequences encoding short numb isoformproteins (and/or numblike), is accompanied or replaced by transient orpermanent transfection/contacting with other proteins and/or nucleicacid sequences, including ones selected from those encoding muscle typespecific bHLH-encoding sequences, MyoD, Myogenin, Myf5, Myf6, Mef2,Myocardin, Ifrdl, Gata 4, Gata 5, and Gata 6.

In another preferred embodiment, when a cardiac muscle cell populationis the desired population, the cells are transfected/contacted withnucleotide sequences including ones selected from those sequencesencoding Myocardin, Gata 4, Gata 5, and Gata 6.

In a preferred embodiment, when a smooth muscle cell population is thedesired cell population, transfection/contacting with short numb isoform(and/or numblike) proteins or with sequences encoding short numb isoformproteins (and/or numblike), is accompanied or replaced by transient orpermanent transfection/contacting with other proteins and/or nucleicacid sequences, including ones selected from those encoding the muscletype specific Myocardin nucleotide sequence.

In a preferred embodiment, when a skeletal muscle cell population is thedesired cell population, transfection/contacting with short numb isoform(and/or numblike) proteins or with sequences encoding short numb isoformproteins (and/or numblike), is accompanied or replaced by transient orpermanent transfection/contacting with other proteins and/or nucleicacid sequences, including ones selected from those encoding the muscletype specific MyoD and myogenin nucleotide sequences.

In a preferred embodiment, when an oligodendrocyte cell population isthe desired cell population, transfection/contacting with short numbisoform (and/or numblike) proteins or with sequences encoding short numbisoform proteins (and/or numblike), is accompanied or replaced bytransient or permanent transfection/contacting with other proteinsand/or nucleic acid sequences, including ones selected from thoseencoding the oligodendrocyte-specific OLIG1, OLIG2, and Zfp488nucleotide sequences.

Simultaneous transfection with any subset of these distinct transgenesequences listed above can be accomplished by any means known to the artincluding the use of multiple genetic vectors, serial transfection aswell as selection based on distinct marker proteins and/or antibioticresistance.

When the desired cell population is a hematopoietic cell population, thedifferentiation medium includes specific agents at concentrationspromoting differentiation into hematopoietic progenitor cells (e.g.vascular endothelial growth factor (VEGF), thrombopoietin, etc. (e.g.Ohmizono, 1997; Wang et al., 2005; Srivastava et al., 2007; Gupta etal., 2007) or differentiated hematopoietic cell types (according tomethods known to the art for providing differentiated hematopoietic celltypes from undifferentiated or pluripotent cells).

When the desired cell population is a germ cell population, thedifferentiation medium includes specific agents at concentrationspromoting differentiation into germ cells (e.g. Nayernia et al. 2006a,2006b).

In a preferred embodiment, when a germ cell population is the desiredcell population, transfection/contacting with short numb isoform (and/ornumblike) proteins or with sequences encoding short numb isoformproteins (and/or numblike), is accompanied or replaced by transient orpermanent transfection/contacting with other proteins and/or nucleicacid sequences, including ones selected from those encoding FIGLA (NCBIaccession No: AY541030 NM_001004311, SEQ ID NO: 130), FIG alpha (NCBIaccession No: U91840, SEQ ID NO: 131), DAZL (NCBI accession No:NM_001190811, SEQ ID NO: 122; NCBI accession No: NM_001351, SEQ ID NO:123), STRA8 (NCBI accession No: NM_182489, SEQ ID NO: 215), FOXL2 (NCBIaccession No: AF522275, SEQ ID NO: 132), OOGENESIN1 (NCBI accession No:NM_178657 XM_622900, SEQ ID NO: 181), OOGENESIN2 (NCBI accession No:NM_198661 XM_355532, SEQ ID NO: 182), OOGENESIN3 (NCBI accession No:NM_201258 XM_131812, SEQ ID NO: 183), OOGENESIN4 (NCBI accession No:NM_001347238 XM_006538858, SEQ ID NO: 184; NCBI accession No: NM_173773,SEQ ID NO: 185), SYCP2 (NCBI accession No: NM_014258 XM_005260247, SEQID NO: 216), SYCP3 (NCBI accession No: NM_001177949, SEQ ID NO: 217;NCBI accession No: NM_153694, SEQ ID NO: 218; NCBI accession No:NM_001177948, SEQ ID NO: 219), SPO11 (NCBI accession No: AF169385, SEQID NO: 207), REC8 (NCBI accession No: NM_005132, SEQ ID NO: 194; NCBIaccession No: NM_001048205, SEQ ID NO: 195), DMC1 (NCBI accession No:NM_007068, SEQ ID NO: 124; NCBI accession No: NM_001363017, SEQ ID NO:125; NCBI accession No: NM_001278208, SEQ ID NO: 126), MOS (NCBIaccession No: NM_005372, SEQ ID NO: 166), STAG3 (NCBI accession No:NM_012447, SEQ ID NO: 212; NCBI accession No: NM_001282716, SEQ ID NO:213; NCBI accession No: NM_001282717, SEQ ID NO: 214), CCNB1 (NCBIaccession No: NM_001354845, SEQ ID NO: 117; NCBI accession No:NM_031966, SEQ ID NO: 118; NCBI accession No: NM_001354844, SEQ ID NO:119), FOXO1 (NCBI accession No: NM_002015, SEQ ID NO: 133), FOXO3 (NCBIaccession No: BC068552, SEQ ID NO: 134), SOHLH1 (NCBI accession No:NM_001101677, SEQ ID NO: 199; NCBI accession No: NM_001012415, SEQ IDNO: 200), SOHLH2 (NCBI accession No: NM_017826 XM_370720, SEQ ID NO:201; NCBI accession No: NM_001282147, SEQ ID NO: 202), NOBOX (NCBIaccession No: NM_001080413, SEQ ID NO: 167; NCBI accession No:XM_017011742, SEQ ID NO: 168), OBOX1 (NCBI accession No: NM_027802, SEQID NO: 173), OBOX2 (NCBI accession No: NM_145708, SEQ ID NO: 174), OBOX3(NCBI accession No: NM_145707, SEQ ID NO: 175), OBOX4 (NCBI accessionNo: AF461109, SEQ ID NO: 176), OBOX6 (NCBI accession No: NM_145710, SEQID NO: 177), LHX8 (NCBI accession No: NM_001001933, SEQ ID NO: 154),LHX9 (NCBI accession No: NM_020204, SEQ ID NO: 155; NCBI accession No:NM_001014434, SEQ ID NO: 156), OOG1 (NCBI accession No: NM_178657XM_622900, SEQ ID NO: 181), SP1 (NCBI accession No: NM_138473 XM_028606,SEQ ID NO: 208; NCBI accession No: NM_003109, SEQ ID NO: 209), ZFP38(NCBI accession No: NM_011757, SEQ ID NO: 232; NCBI accession No:NM_001044703, SEQ ID NO: 233; NCBI accession No: NM_001044704, SEQ IDNO: 234), TRF2 (NCBI accession No: U95970, SEQ ID NO: 229), TB2/TRF3(NCBI accession No: AY457923, SEQ ID NO: 230), TAF4B (NCBI accession No:NM_001293725, SEQ ID NO: 220; NCBI accession No: NM_005640, SEQ ID NO:221), TAF7L (NCBI accession No: NM_024885, SEQ ID NO: 222), TAF71 (NCBIaccession No: NM_001168474, SEQ ID NO: 223), TIA1 (NCBI accession No:NM_022037, SEQ ID NO: 225; NCBI accession No: NM_022173, SEQ ID NO: 226;NCBI accession No: NM_001351508, SEQ ID NO: 227), PHTF1 (NCBI accessionNo: NM_006608, SEQ ID NO: 189; NCBI accession No: NM_001323041, SEQ IDNO: 190; NCBI accession No: NM_001323042, SEQ ID NO: 191), TNP2 (NCBIaccession No: NM_005425, SEQ ID NO: 228), HILS1 (NCBI accession No:NR_024193, SEQ ID NO: 145), DAZL (NCBI accession No: NM_001190811, SEQID NO: 122; NCBI accession No: NM_001351, SEQ ID NO: 123), BMP15 (NCBIaccession No: NM_005448, SEQ ID NO: 116), PTTG3 (NCBI accession No:NR_002734, SEQ ID NO: 193), AURKC (NCBI accession No: NM_001015878, SEQID NO: 114; NCBI accession No: NM_001015879, SEQ ID NO: 115), OTX2 (NCBIaccession No: NM_021728, SEQ ID NO: 186; NCBI accession No: NM_172337,SEQ ID NO: 187; NCBI accession No: NM_001270523, SEQ ID NO: 188), SOX15(NCBI accession No: NM_006942, SEQ ID NO: 203), SOX30 (NCBI accessionNo: NM_178424, SEQ ID NO: 204; NCBI accession No: NM_007017, SEQ ID NO:205; NCBI accession No: NM_001308165, SEQ ID NO: 206), FOXR1 (NCBIaccession No: NM_181721, SEQ ID NO: 135), ALF (NCBI accession No:NM_001133, SEQ ID NO: 113), OCT4 (NCBI accession No: NM_002701, SEQ IDNO: 178; NCBI accession No: NM_203289, SEQ ID NO: 179; NCBI accessionNo: NM_001173531, SEQ ID NO: 180), DPPA3/STELLA (NCBI accession No:BC062480, SEQ ID NO: 129), ZFP38 (NCBI accession No: NM_011757, SEQ IDNO: 232; NCBI accession No: NM_001044703, SEQ ID NO: 233; NCBI accessionNo: NM_001044704, SEQ ID NO: 234), RPS6KA3 (NCBI accession No: NM_004586XM_939339 XM_944112, SEQ ID NO: 196), HINFP (NCBI accession No:NM_015517, SEQ ID NO: 141; NCBI accession No: NM_198971, SEQ ID NO: 142;NCBI accession No: NM_001243259, SEQ ID NO: 143; NCBI accession No:NM_001351957, SEQ ID NO: 144), NPAT (NCBI accession No: D83243, SEQ IDNO: 169), SP1 (NCBI accession No: NM_138473 XM_028606, SEQ ID NO: 208;NCBI accession No: NM_003109, SEQ ID NO: 209), SP3 (NCBI accession No:NM_003111 XM_092672, SEQ ID NO: 210; NCBI accession No: NM_001017371,SEQ ID NO: 211), HOXA1 (NCBI accession No: NM_005522, SEQ ID NO: 146;NCBI accession No: NM_153620, SEQ ID NO: 147), HOXA7 (NCBI accession No:AJ005814, SEQ ID NO: 148), HEX (NCBI accession No: Z21533, SEQ ID NO:140), YP30 (NCBI accession No: NM_214493, SEQ ID NO: 231), ZP1 (NCBIaccession No: NM_207341, SEQ ID NO: 235), ZP2 (NCBI accession No:NM_003460, SEQ ID NO: 236; NCBI accession No: NM_001290104, SEQ ID NO:237), ZP3 (NCBI accession No: X56777 S53912, SEQ ID NO: 238), SFE1 (NCBIaccession No: NM_001170815, SEQ ID NO: 197), SFE9 (NCBI accession No:AY540956, SEQ ID NO: 198), OPO, PLN (NCBI accession No: NM_002667, SEQID NO: 192), RDV, GLD1, MMU-MiR351 (NCBI accession No: NR_029776, SEQ IDNO: 161), MMU-MiR615 (NCBI accession No: NR_030526, SEQ ID NO: 164),MMU-MiR592 (NCBI accession No: NR_030420, SEQ ID NO: 163), MMU-MiR882(NCBI accession No: NR_030540, SEQ ID NO: 165), MMU-MiR185 (NCBIaccession No: NR_029571, SEQ ID NO: 157), MMU-MiR491 (NCBI accession No:NR_030478, SEQ ID NO: 162), MMU-MiR326 (NCBI accession No: NR_029891,SEQ ID NO: 159), MMU-MiR330 (NCBI accession No: NR_029763, SEQ ID NO:160), MMU-MiR351 (NCBI accession No: NR_029776, SEQ ID NO: 161).

For example, but not limiting, in one preferred embodiment, when a spermor spermatocyte cell population is the desired cell population,transfection/contacting with short numb isoform (and/or numblike)proteins or with sequences encoding short numb isoform proteins (and/ornumblike), is accompanied or replaced by transient or permanenttransfection/contacting with other proteins and/or nucleic acidsequences, including ones selected from those encoding SYCP2 (NCBIaccession No: NM_014258 XM_005260247, SEQ ID NO: 216), SYCP3 (NCBIaccession No: NM_001177949, SEQ ID NO: 217; NCBI accession No:NM_153694, SEQ ID NO: 218; NCBI accession No: NM_001177948, SEQ ID NO:219), SPO11 (NCBI accession No: AF169385, SEQ ID NO: 207), REC8 (NCBIaccession No: NM_005132, SEQ ID NO: 194; NCBI accession No:NM_001048205, SEQ ID NO: 195), DMC1 (NCBI accession No: NM_007068, SEQID NO: 124; NCBI accession No: NM_001363017, SEQ ID NO: 125; NCBIaccession No: NM_001278208, SEQ ID NO: 126), MOS (NCBI accession No:NM_005372, SEQ ID NO: 166), STAG3 (NCBI accession No: NM_012447, SEQ IDNO: 212; NCBI accession No: NM_001282716, SEQ ID NO: 213; NCBI accessionNo: NM_001282717, SEQ ID NO: 214), OCT4 (NCBI accession No: NM_002701,SEQ ID NO: 178; NCBI accession No: NM_203289, SEQ ID NO: 179; NCBIaccession No: NM_001173531, SEQ ID NO: 180), ALF (NCBI accession No:NM_001133, SEQ ID NO: 113), RPS6KA3 (NCBI accession No: NM_004586XM_939339 XM_944112, SEQ ID NO: 196), HINFP (NCBI accession No:NM_015517, SEQ ID NO: 141; NCBI accession No: NM_198971, SEQ ID NO: 142;NCBI accession No: NM_001243259, SEQ ID NO: 143; NCBI accession No:NM_001351957, SEQ ID NO: 144), SP1 (NCBI accession No: NM_138473XM_028606, SEQ ID NO: 208; NCBI accession No: NM_003109, SEQ ID NO:209), SP3 (NCBI accession No: NM_003111 XM_092672, SEQ ID NO: 210; NCBIaccession No: NM_001017371, SEQ ID NO: 211), TAF71 (NCBI accession No:NM_001168474, SEQ ID NO: 223), TIA1 (NCBI accession No: NM_022037, SEQID NO: 225; NCBI accession No: NM_022173, SEQ ID NO: 226; NCBI accessionNo: NM_001351508, SEQ ID NO: 227), PHTF1 (NCBI accession No: NM_006608,SEQ ID NO: 189; NCBI accession No: NM_001323041, SEQ ID NO: 190; NCBIaccession No: NM_001323042, SEQ ID NO: 191), TNP2 (NCBI accession No:NM_005425, SEQ ID NO: 228), HILS1 (NCBI accession No: NR_024193, SEQ IDNO: 145), CLGN (NCBI accession No: NM_001130675, SEQ ID NO: 120; NCBIaccession No: NM_004362, SEQ ID NO: 121), TEKT1 (NCBI accession No:NM_053285, SEQ ID NO: 224), FSCN3 (NCBI accession No: NM_020369, SEQ IDNO: 136), DNAHC8 (NCBI accession No: NM_001206927, SEQ ID NO: 127; NCBIaccession No: NM_001371, SEQ ID NO: 128), LDHC (NCBI accession No:NM_017448, SEQ ID NO: 149; NCBI accession No: NM_002301, SEQ ID NO:150), ADAM3 (NCBI accession No: AK302269, SEQ ID NO: 110), OAZ3 (NCBIaccession No: NM_016178, SEQ ID NO: 170; NCBI accession No:NM_001134939, SEQ ID NO: 171; NCBI accession No: NM_001301371, SEQ IDNO: 172), AKAP3 (NCBI accession No: NM_001278309, SEQ ID NO: 111; NCBIaccession No: NM_006422, SEQ ID NO: 112), MMU-MiR351 (NCBI accession No:NR_029776, SEQ ID NO: 161), MMU-MiR615 (NCBI accession No: NR_030526,SEQ ID NO: 164), MMU-MiR592 (NCBI accession No: NR_030420, SEQ ID NO:163), MMU-MiR882 (NCBI accession No: NR_030540, SEQ ID NO: 165), andMMU-MiR185 (NCBI accession No: NR_029571, SEQ ID NO: 157).

For example, but not limiting, in one preferred embodiment, when aoocyte cell population is the desired cell population,transfection/contacting with short numb isoform (and/or numblike)proteins or with sequences encoding short numb isoform proteins (and/ornumblike), is accompanied or replaced by transient or permanenttransfection/contacting with other proteins and/or nucleic acidsequences, including ones selected from those encoding MOS (NCBIaccession No: NM_005372, SEQ ID NO: 166), CCNB1 (NCBI accession No:NM_001354845, SEQ ID NO: 117; NCBI accession No: NM_031966, SEQ ID NO:118; NCBI accession No: NM_001354844, SEQ ID NO: 119), OCT4 (NCBIaccession No: NM_002701, SEQ ID NO: 178; NCBI accession No: NM_203289,SEQ ID NO: 179; NCBI accession No: NM_001173531, SEQ ID NO: 180), FIGalpha (NCBI accession No: U91840, SEQ ID NO: 131), FIGL alpha (NCBIaccession No: AY541030 NM_001004311, SEQ ID NO: 130), ALF (NCBIaccession No: NM_001133, SEQ ID NO: 113), SOHLH1 (NCBI accession No:NM_001101677, SEQ ID NO: 199; NCBI accession No: NM_001012415, SEQ IDNO: 200), SOHLH2 (NCBI accession No: NM_017826 XM_370720, SEQ ID NO:201; NCBI accession No: NM_001282147, SEQ ID NO: 202), LHX8 (NCBIaccession No: NM_001001933, SEQ ID NO: 154), LHX9 (NCBI accession No:NM_020204, SEQ ID NO: 155; NCBI accession No: NM_001014434, SEQ ID NO:156), OOG1 (NCBI accession No: NM_178657 XM_622900, SEQ ID NO: 181), FIGalpha (NCBI accession No: U91840, SEQ ID NO: 131), SP1 (NCBI accessionNo: NM_138473 XM_028606, SEQ ID NO: 208; NCBI accession No: NM_003109,SEQ ID NO: 209), LHX3 (NCBI accession No: NM_178138, SEQ ID NO: 151;NCBI accession No: NM_014564, SEQ ID NO: 152; NCBI accession No:NM_001363746, SEQ ID NO: 153), LHX9 (NCBI accession No: NM_020204, SEQID NO: 155; NCBI accession No: NM_001014434, SEQ ID NO: 156), TBP2/TRF3(NCBI accession No: AY457923, SEQ ID NO: 230), DAZL (NCBI accession No:NM_001190811, SEQ ID NO: 122; NCBI accession No: NM_001351, SEQ ID NO:123), BMP15 (NCBI accession No: NM_005448, SEQ ID NO: 116), GDF9 (NCBIaccession No: NM_005260, SEQ ID NO: 137; NCBI accession No:NM_001288824, SEQ ID NO: 138; NCBI accession No: NM_001288825, SEQ IDNO: 139), PTTG3 (NCBI accession No: NR_002734, SEQ ID NO: 193), AURKC(NCBI accession No: NM_001015878, SEQ ID NO: 114; NCBI accession No:NM_001015879, SEQ ID NO: 115), OTX2 (NCBI accession No: NM_021728, SEQID NO: 186; NCBI accession No: NM_172337, SEQ ID NO: 187; NCBI accessionNo: NM_001270523, SEQ ID NO: 188), SOX15 (NCBI accession No: NM_006942,SEQ ID NO: 203), SOX30 (NCBI accession No: NM_178424, SEQ ID NO: 204;NCBI accession No: NM_007017, SEQ ID NO: 205; NCBI accession No:NM_001308165, SEQ ID NO: 206), FOXR1 (NCBI accession No: NM_181721, SEQID NO: 135), NOBOX (NCBI accession No: NM_001080413, SEQ ID NO: 167;NCBI accession No: XM_017011742, SEQ ID NO: 168), OBOX1 (NCBI accessionNo: NM_027802, SEQ ID NO: 173), OBOX2 (NCBI accession No: NM_145708, SEQID NO: 174), OBOX3 (NCBI accession No: NM_145707, SEQ ID NO: 175), OBOX6(NCBI accession No: NM_145710, SEQ ID NO: 177), OOGENESIN1 (NCBIaccession No: NM_178657 XM_622900, SEQ ID NO: 181), OOGENESIN2 (NCBIaccession No: NM_198661 XM_355532, SEQ ID NO: 182), OOGENESIN3 (NCBIaccession No: NM_201258 XM_131812, SEQ ID NO: 183), OOGENESIN4 (NCBIaccession No: NM_001347238 XM_006538858, SEQ ID NO: 184; NCBI accessionNo: NM_173773, SEQ ID NO: 185), YP30 (NCBI accession No: NM_214493, SEQID NO: 231), ZP1 (NCBI accession No: NM_207341, SEQ ID NO: 235), ZP2(NCBI accession No: NM_003460, SEQ ID NO: 236; NCBI accession No:NM_001290104, SEQ ID NO: 237), ZP3 (NCBI accession No: X56777 S53912,SEQ ID NO: 238), SFE1 (NCBI accession No: NM_001170815, SEQ ID NO: 197),SFE9 (NCBI accession No: AY540956, SEQ ID NO: 198), OPO, PLN (NCBIaccession No: NM_002667, SEQ ID NO: 192), RDV, GLD1, DAZL (NCBIaccession No: NM_001190811, SEQ ID NO: 122; NCBI accession No:NM_001351, SEQ ID NO: 123), STRA8 (NCBI accession No: NM_182489, SEQ IDNO: 215), MMU-MiR615 (NCBI accession No: NR_030526, SEQ ID NO: 164),MMU-MiR491 (NCBI accession No: NR_030478, SEQ ID NO: 162), MMU-MiR326(NCBI accession No: NR_029891, SEQ ID NO: 159), MMU-MiR330 (NCBIaccession No: NR_029763, SEQ ID NO: 160), MMU-MiR212 (NCBI accession No:NR_029794, SEQ ID NO: 158) and MMU-MiR351 (NCBI accession No: NR_029776,SEQ ID NO: 161).

When the desired cell population is an endoderm and pancreatic isletcell population, the differentiation media includes specific agents atconcentrations promoting differentiation into endoderm and pancreaticislet cells (e.g. Xu et al., 2006; Denner et al., 2007; Shim et al.,2007; Jiang et al., 2007).

In a preferred embodiment, differentiation of selected cells and/ortheir progeny may occur in the differentiation medium in the absence oftransfection with numblike, short Numb isoforms or othertransgenes/proteins, although the differentiation medium may beunchanged.

In embodiments, a single vector will be utilized which controls theexpression of nucleotide sequence(s) encoding the “long” (PRR+)isoform(s) of the mammalian numb gene (and/or synthetic oligonucleotidestargeting numblike or the short numb isoforms) under one regulablepromoter (e.g. a tetracyc line-regulated promoter), while the Numblikeand short Numb isoforms (and/or synthetic oligonucleotides targeting thelong (PRR+) isoforms) are expressed under the control of another,distinct, but also regulable promoter. Thus, the long (PRR+) numbisoform(s) can be expressed (and/or short isoforms repressed) whenexpansion of the selected cells is desired and an inducing agent (e.g.tetracycline) is added to the growth medium; later numblike and theshort isoforms can be expressed (and/or long (PRR+) numb isoform(s)repressed) when differentiation is desired.

Alternatively, proteins and peptides corresponding to Numb isoforms,Notch, OCT3/4, SOX2, and other DNA sequences listed herein may beapplied in analogous fashion to selected cells and/or their progeny viaelectroporation (e.g. Koken et al., 1994; Ritchie and Gilroy, 1998),using nano particles, cationic lipids, fusogenic liposomes (e.g.Yoshikawa et al., 2005; 2007), etc. in lieu of, or in combination withgenetic transfection. Generally, electroporation allows for hightransfection efficiency (and efficient production of the desired cells)without genomic integration of the transgene and is therefore associatedwith increased safety.

The DNA or RNA encoding protein(s) or polypeptide(s) promotingproliferation, multipotentiality, pluripotentiality or differentiationof the selected cells may be isolated in accordance with standardgenetic engineering techniques (for example, by isolating such DNA froma cDNA library of the specific cell line) and placing it into anappropriate expression vector, which then is transfected into theselected cells.

In another preferred embodiment, endoderm and pancreatic islet cells arethe desired population, and transfection/contacting with short numbisoform (and/or numblike) proteins or with sequences encoding short numbisoform proteins (and/or numblike), is accompanied or replaced bytransient or permanent transfection/contacting with other proteinsand/or nucleic acid sequences, including ones selected from thoseencoding Foxa2, Sox17, HLXB9 and/or Pdxl.

In another preferred embodiment, hepatocytes are the desired population,and transfection/contacting with short numb isoform (and/or numblike)proteins or with sequences encoding short numb isoform proteins (and/ornumblike), is accompanied or replaced by transient or permanenttransfection/contacting with other proteins and/or nucleic acidsequences, including ones selected from those encoding hepatic nuclearfactor (HNF)-I, IINF-3, IINF-4, HNF-6 and creb-binding protein.

In another preferred embodiment, hematopoietic cells are the desiredpopulation, and transfection/contacting with short numb isoform (and/ornumblike) proteins or with sequences encoding short numb isoformproteins (and/or numblike), is accompanied or replaced by transient orpermanent transfection/contacting with other proteins and/or nucleicacid sequences, including ones selected from those encoding Runxl/AML1and NOV(CCN3), and/or cell culture in the presence of colony stimulatingfactors specific for the desired cell populations. The Runxl/AML1 aisoform is introduced when engraftment is desired and the b isoform whendifferentiation is desired (Creemers et al., 2006).

In another preferred embodiment, chondrocytes are the desiredpopulation, and lransfcction with sequences encoding short numb isoforms(and/or numblike) is accompanied or replaced by transient or permanenttransfection of other sequences including ones encoding Sox9,CREB-binding protein, Gataó and/or Runx2.

In another preferred embodiment, bone cells (especially osteoblasts) arethe desired population, and transfection with sequences encoding shortnumb isoforms (and/or numblike) is accompanied or replaced by transientor permanent transfection of other sequences including Runx2.

In a preferred embodiment, the genetic vectors encoding the long Numbisoforms (such as those described herein) are introduced transiently orunder the control of a regulable promoter, into endogenous cells in vivoin order to cause those cells proliferate transiently.

In a preferred embodiment, endogenous cells (e.g. ependymal zone cellsof the central nervous system) are transfected/contacted in vivo withgenetic vectors encoding either the shortest numb isoform or thenumblike protein(s) alone or in conduction with othertransgenes/proteins named herein, in order to transiently or permanentlypromote renewed or increased differentiation (especially neuronaldifferentiation) and migration of progenitor/ependymal cells in thecentral nervous system). This renewal or increase is measured in termsof the number of cells showing new-onset expression of markersassociated with differentiation. This may be accomplished byintroduction of the genetic vectors into the organ system using methodssuitable for that purpose (see examples).

In a preferred embodiment, endogenous cells (e.g. ependymal zone cellsof the central nervous system) are transfected/contacted in vivo withgenetic vectors encoding the long numb isoform(s) and/or othertransgenes/proteins named herein, in order to transiently promoterenewed or increased stem cell proliferation (with subsequentdifferentiation of progeny cells). This renewal or increase is measuredin terms of the number of cells showing new-onset expression of marlersassociated with dividing progenitors. This may be accomplished byintroduction of the genetic vectors into the organ system using methodssuitable for that purpose (see examples).

Likewise this approach is also be suitable for inducing renewed orincreased differentiation from other stem cell populations in othertissues (such as the skin, etc). This approach can be utilized, forexample, clinically in the setting of central nervous system injury,disorders of other tissues where normal differentiation or migration areinadequate, dysplaslic disorders and other disorders where the approachis beneficial.

In a preferred embodiment, nucleic acid(s) or protein(s) correspondingto a single gene, or portion thereof, (particularly those named herein,discovered according to methods described herein, discovered accordingto other published methods; and/or known to be capable of initiating thedesired manner of differentiation) are the only nucleic acid(s) orprotein(s) overexpressed and/or introduced to initiate differentiationin the selected cells.

In a preferred embodiment, nucleic acid(s) or protein(s) correspondingto a single gene, or portion thereof, (particularly those named herein,discovered according to methods described herein, discovered accordingto other published methods; and/or known to be capable of initiating thedesired manner of differentiation) are the only nucleic acid(s) orprotein(s) overexpressed and/or introduced to initiate differentiationin the selected cells and the method utilized is electroporation,liposomes, nanocapsules, nanovaults, and/or another approach avoidingretroviral/lentiviral integration or other random alteration of thecell's genome.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)can be utilized in concert with the nucleic acid(s) or protein(s)corresponding to a single gene, or portion thereof, (particularly thosenamed herein, discovered according to methods described herein,discovered according to other published methods; and/or known to becapable of initiating the desirable manner of differentiation) so longas a population of differentiating cells is produced from the selectedcells.

In a separate preferred embodiment, other nucleic acid(s) or protein(s)can be utilized in concert with the nucleic acid(s) or protein(s)corresponding to a single gene, or portion thereof, (particularly thosenamed herein, discovered according to methods described herein,discovered according to other published methods; and/or known to becapable of initiating the desirable manner of differentiation) so longas a population of differentiating cells is produced from the selectedcells and the method utilized is electroporation, liposomes,nanocapsules, nanovaults, and/or another approach avoidingretroviral/lentiviral integration or other random alteration of thecell's genome.

It is to be understood that any combination of nucleic acid or proteinsequences described herein can be modified by excluding thosecorresponding to Numb and/or Numblike so long as the desired cellpopulation or behavior is achieved.

Similarly, it should be understood that the methods described herein (orelsewhere) for initiating differentiation are applicable to any inducedor non-induced multipotent, pluripotent, or self-renewing stem cells, orother selected cells, not only those obtained in the manner describedherein.

Sources of Selected Cells

The population of selected cells may derive from various stem cells,progenitor cells and somatic cells. However somatic cells lacking nuclei(e.g. mature, human red blood cells) are specifically excluded. Selectedstem cells may be derived from existing cell lines or isolated fromstored, banked, or cryopreserved sources. Typical sources of stem cellsinclude bone marrow, peripheral blood, placental blood, amniotic fluid(e.g. De Coppi et al., 2007), umbilical cord blood (e.g. Zhao, et al.,2006; Tian et al., 2007), adipose tissue (e.g. Gimble et al., 2007; Maet al., 2007), non-human embryos, and others. Circulating leukocytes andother non-stem cells may likewise be selected and subjected to the sameculture conditions as described above effective that they acquiremultipotency, pluripotency and/or self-renewal as a result. Examples ofother accessible somatic cells useful in this invention includelymphocytes and epithelial (e.g. buccal cheek) cells. Isolation andcollection of cells selected for use within the present invention may beperformed by any method known to the art.

In embodiments involving animals, stem cells isolated from prostate,testis, embryonic brain, and intestine are also disclosed as beingpreferred sources of selected cells.

In a preferred embodiment, the selected cells and/or their progeny arecultured in a three-dimensional format.

A further aim of the present invention is to provide cells for use inthe production of patient-compatible and patient-specific tissues andorgans for transplantation to patients deemed to be requiring suchorgans or tissues. It is disclosed herein that the pluripotent,multipotent, and/or differentiating cells provided by the methodsdescribed herein (or similar methods) be utilized in conjunction withtechniques aimed at the production of such organs and/or tissues (e.g.Boland et al., 2006. Xu et al., 2006; Campbell and Weiss, 2007). Suchutilization is specifically covered by the present invention.

For instance, pluripotent, multipotent, and/or differentiating cellsproduced or treated according to the methods desribed herein (or otherpublished methods) may be grown in association with three-dimcsnisonalor two-dimensional scaffoldings engineered to replicate normal tissuestructure and/or organ structures (e.g. Yarlagada et al., 2005; Kim etal, 1998; WO/2003/070084; EP1482871; WO03070084; U.S. Pat. Nos.2,395,698; 7,297,540; 6,995,013; 6,800,753; Isenberg et al., 2006).

Similarly, scaffoldings to be occupied by the pluripotent, multipotent,and/or differentiating cells may be derived from cadaveric organ(s) ortissue(s) after the cadaveric organs or tissues (e.g. bone, heart,kidney, liver, lung, etc.) may be treated in such away that the hostimmune cells resident in that tissue, and other undesirable or ancillaryhost cells, are eliminated (e.g. by ionizing radiation, sterilization(e.g. Mroz et al., 2006), and/or various methods of decellularization(U.S. Pat. Nos. 6,734,018; 6,962,814; 6,479,064; 6,376,244; 5,032,508;4,902,508; 4,956,178; 5,281,422, 5,554,389; 6,099,567; and 6,206,931;4361552 and 6576618; 6753181; U.S. application Ser. No. 11/162,715;WO/2001/048153; WO/2002/024244; WO003002165; WO/2001/049210;WO/2007/025233; European Patents EP1482871; EP1246903; EP1244396;EP0987998; EP1244396; EP1 333870; Rieder et al., 2004; Ott et al., 2008;Taylor et al., 1998)).

Likewise, it is anticipated that the pluripotent, multipotent, and/ordifferentiating cells of the present invention may be used inapplications utilizing inkjet-style printing for tissue engineering(e.g. Boland et al., 2006. Xu et al., 2006; Campbell et al., 2007).

Therefore such use of the cells produced or treated according to themethods described herein is covered.

In another preferred embodiment, the selected cells and/or their progenyare cultured in hanging drops.

In accordance with another aspect of the present invention, selectedcells may be modified genetically beforehand.

In accordance with another aspect of the present invention, selectedcells may be modified with DNA or RNA encoding protein(s) orpolypeptide(s) promoting differentiation of the cell into a desired cellpopulation.

Screening Cell Populations

In one embodiment, the methods of this invention comprise screeningcells from cell lines, donor sources, umbilical cord blood, andautologous or donor bone marrow, blood, spermatogonia, primordial germcells, buccal cheek cells, or any other cell source effective in thecurrent invention. Selected cells can be screened to confirm successfultransfection with beneficial sequence(s) or therapeutic vector(s) aswell as successful initiation of differentiation by any method known tothe art (Guan et al., 2006; U.S. Pat. No. 6,432,711). In someembodiments, the cells are screened using standard PCR and nucleic acidhybridization-based methods or using rapid typing methods. In preferredembodiments, the cells are screened according to expression of reportergenes. In some embodiments, cells are screened by expression of a markergene encoded by the transgene expressing vector(s) such as an antibioticresistance gene or a fluorescent protein (e.g. GFP) gene.

Screening for Therapeutic Vectors and Beneficial Sequences

Cells can be screened for the presence of beneficial sequence(s) andtherapeutic vector(s) using any method(s) known to the art for detectionof specific sequences. Each cell sample can be screened for a variety ofsequences simultaneously. Alternatively, multiple samples can bescreened simultaneously.

Cell differentiation may be monitored by several means: including (i)morphological assessment, (ii) utilizing reverse transcriptasepolymerase chain reaction (RT-PCR), Northern blot, or microarraytechniques to monitor changes in gene expression, (iii) assayingcellular expression of specific markers such as beta tubulin III (forneurons) etc. (Ozawa, et al., 1985). In some embodiments, the cells arescreened for successful initiation of differentiation using FACS sortingbased on cell type specific markers or transgenic marker expression(e.g. antibiotic resistance or fluorescent protein expression) under thecontrol of cell type specific promoters such as the myosin promoter inmuscle cells; the human cardiac a-actin promoter in cardiomyocytes; theinsulin promoter in insulin producing cells; the neuronal-specificenolase (NSE) promoter for neuronal differentiation, or neurotransmitterrelated promoters such as the tyrosine hydroxylase promoter indopaminergic neurons; etc.). In some embodiments, the cells are screenedusing standard PCR and nucleic acid hybridization-based methods. In aparticularly preferred embodiment, the cells are screened using rapidtyping methods.

Screening for Human Leukocyte Antigen (HLA) Type

In certain embodiments, the selected cells are selected with respect tocompatible HLA typing. The HLA genotype can be determined by any meansknown to those of skill in the art.

The cells used for screening may consist of cells taken directly from adonor, or from cell lines established from donor cells, or otherpracticable cell sources. The cells can be screened for beneficialsequence(s), and/or therapeutic vector(s) and HLA type at once, orseparately. Those cells successfully transfected with a beneficialsequence and showing an appropriate HLA genotype can be prepared fortransplantation to a patient.

In certain embodiments, the transfected/contacted cells are transplantedwithout HLA typing. In other embodiments, the cells are HLA typed forcompatibility.

Screening for Agents Promoting a Cellular Phenotype

The present invention also provides for methods of screening proteinsand agents for their ability to induce phenotypic changes ordifferentiation of the selected cells and/or their progeny into desiredcell populations. Briefly, vectors encoding complementary DNAs (cDNAs)from appropriate cDNA libraries are transfected into the selectedcells/and or their progeny. Once a specific cDNA that inducesdifferentiation or other phenotypic change is identified, such cDNA thenmay be isolated and cloned into an appropriate expression vector forprotein production in appropriate cells (e.g. COS cells) in vitro. Laterthe protein containing supernatant can be applied to the selected cellcultures to determine if any secreted proteins from such cells inducedifferentiation Alternatively, candidate agents can be applied to theselected cell cultures to determine if any secreted proteins from suchcells induce differentiation (see U.S. Pat. No. 6,432,711).

The present invention also provides for methods of screening nucleicacids for their ability to induce multipotentiality, pluripotentiality,and/or self-renewal, or to initiate differentiation of selected cellsand/or their progeny. In these methods, vectors encoding selected cDNAs(or cDNAs from appropriate cDNA libraries, or other sequences) areintroduced into the selected cells/and or their progeny usingelectroporation, nanocapsules, nanovaults, liposomes, retroviruses,lentiviruses, and/or any other practicable means of transfection. Once aspecific cDNA that induces a phenotypic change, multipotentiality,pluripotentiality, and/or self-renewal, is identified, such cDNA thenmay be isolated and cloned into an appropriate expression vector. Assaysfor determining such changes include those described elsewhere herein.

Likewise the protein corresponding to the identified cDNA may beproduced in appropriate cells (e.g. COS cells) in vitro to determinewhether the protein containing supernatant can be applied to theselected cell cultures and induce the desired changes.

Finally, proteins may be introduced into the selected cells/and or theirprogeny using electroporation, nanocapsules, nanovaults, liposomes,retroviruses, lentiviruses, and/or any other practicable means oftransfection, and the resulting cells assessed as described herein formultipotentiality, pluripotentiality, self-renewal or the initiation ofdifferentiation.

Tranplantation of Cells into Patients

After screening, selected cells and/or their progeny may becryopreserved, maintained as cell lines in culture, or may beadministered to the patient. Selected cells can be cryopreserved ormaintained in culture by any means known to the art and preserved forfuture transplantation procedures.

Preferably, the cells to be screened are obtained from accessiblesources allowing easy collection.

With regard to producing HIV resistant cells: targeted somatic cells andstem cells of this invention can be of any type capable ofdifferentiating into cells that can be infected by HIV, that can sustainthe transcription and/or replication of HIV, that can alter the HIVimmune response, or that can retard progression to AIDS. Such stem cellsinclude, but are not limited to, pluripotent cells derived fromspermatogonia, primordial germ cells, hematopoietic stem cells,peripheral blood cells, placental blood cells, amniotic fluid cells,umbilical cord blood cells, buccal cheek cells, adipose tissue cells(including stem cells derived from those tissues), reprogrammed cells,induced multipotent cells, induced pluripotent cells, etc., non-humanembryos, and/or any other cell type that can form blood and immunecells, HIV target cells, and other cells.

Therapeutic vector(s) express “beneficial sequence(s)” intended torender transfected/contacted or infected cells less capable ofsustaining HIV replication and transcription. The genetic vectorexpressing “beneficial sequence(s)” as well as any virus derived fromsuch genetic vector, are herein termed “therapeutic vector”.

After screening, cells transfected/contacted with the desiredtherapeutic vector(s) and expressing beneficial sequence (with orwithout compatible HLA genotype) may be expanded ex vivo (in vitro)using standard methods to culture dividing cells and maintained asstable cell lines (U.S. Pat. Nos. 6,432,711 and 5,453,357 hereinincorporated by reference). Alternatively, these cells can beadministered to the patient and expanded in vivo.

Selected cells can be cryopreserved by any means known to the art andpreserved for future transplantation procedures.

Transplantation of Desirable Cell Populations into Patients

In certain embodiments, cell populations are enriched for stem cellsprior to transplantation. Various methods to select for stem cells arewell known in the art. For example, cell samples can be enriched byfluorescently labeled monoclonal antibodies recognizing cell-surfacemarkers of undifferentiated hematopoietic stem cells (e.g., CD34, CD59,Thyl, CD38 low, C-kit low, lin− minus) for sorting viafluorescence-activated cell sorting (FACS).

In other embodiments, a sample of the selected cells is transplanted,without enrichment.

In some embodiments, the endogenous stem cells of the bone marrow areeliminated or reduced prior to transplantation of the therapeutic stemcells. Therapeutic stem cells are defined as those stem cells containingbeneficial sequence(s) or therapeutic vector(s).

In some embodiments, the transplantation process may involve thefollowing phases: (1) conditioning, (2) stem cell infusion, (3)neutropenic phase, (4) engraftment phase, and (5) post-engraftmentperiod.

In some embodiments, the endogenous stem cells that normally produce thedesired cells (e.g. bone marrow stem cells) are eliminated or reducedprior to transplantation.

Chemotherapy, radiation, etc. and/or methods analogous to thosedescribed in U.S. Pat. No. 6,217,867 may be used to condition the bonemarrow for appropriate engraftment of the transplant. Finally,therapeutic stem cells may be transplanted into the patient using anymethod known to the art.

Design of Numb/Numblike and Other Transgene Encoding Vectors

In one embodiment transfection with nucleic acid sequence(s) encodingnumblike/numb isoform(s) is accomplished via viral transfection. Theterm “‘Numb/Numblike encoding vector(s)” refers to the vectorsincorporating the nucleic acid sequence(s) encoding numblike/numbisoform(s) and/or synthetic oligonucleotides targeting numblike or numbisoforms, as well as any additional transgene sequences, syntheticoligonucleoties, etc, and any associated viral supernatant incorporatedin those vector sequences.

The Numb/Numblike Encoding Vector(s) May Comprise an Expression Vector.

Appropriate expression vectors are those that may be employed fortransfecting DNA or RNA into eukaryotic cells. Such vectors include, butare not limited to, prokaryotic vectors such as, for example, bacterialvectors; eukaryotic vectors, such as, for example, yeast vectors andfungal vectors; and viral vectors, such as, but not limited toadenoviral vectors, adeno-associated viral vectors, and retroviralvectors. Examples of retroviral vectors which may be employed include,but are not limited to, those derived from Moloney Murine LeukemiaVirus, Moloney Murine Sarcoma Virus, and Rous Sarcoma Virus, FIV, HIV,SIV and hybrid vectors.

It is disclosed that the Numb/Numblike encoding vector(s) may be used totransfect cells in vitro and/or in vivo. Transfection can be carried outby any means known to the art, especially through virus produced fromviral packaging cells. Such virus may be encapsidated so as to becapable of infecting a variety of cell types. Nevertheless, anyencapsidation technique allowing infection of selected cell types and/ortheir progeny is practicable within the context of the presentinvention.

Design of Human Immunodeficiency Virus (HIV) Gene Therapy Vector(s)

The “therapeutic vector(s)” may incorporate an expression vector.Appropriate expression vectors are those that may be employed fortransfecting DNA or RNA into eukaryotic cells. Such vectors include, butare not limited to, prokaryotic vectors such as, for example, bacterialvectors; eukaryotic vectors, such as, for example, yeast vectors andfungal vectors; and viral vectors, such as, but not limited toadenoviral vectors, adeno-associated viral vectors, and retroviralvectors. Examples of retroviral vectors which may be employed include,but are not limited to, those derived from Moloney Murine LeukemiaVirus, Moloney Murine Sarcoma Virus, and Rous Sarcoma Virus, felineimmunodeficiency virus (FIV), HIV, simian immunodeficiency virus (SIV)and hybrid vectors.

It is disclosed herein that the therapeutic vector(s) may be used totransfect target cells in vitro and/or in vivo. Transfection can becarried out by any means known to the art, especially through virusproduced from viral packaging cells. Such virus may be encapsidated soas to be capable of infecting CD34+ cells and/or CD4+ cells. However, insome instances, other cell types are transfected/contacted by means notinvolving the CD4 or CD34 proteins. Nevertheless, any encapsidationtechnique allowing infection of such cell types may therefore beincluded in the disclosure of the present invention.

Pseudotyping with different envelope proteins expands the range of hostcells transduceable by viral vectors and therapeutic vectors, and allowsthe virus to be concentrated to high titers, especially when pseudotypedwith the vesicular stomatitis virus envelope glycoprotein (VSV-G) (Li etal., 1998; Reiser et al., 2000).

Vector Construction

Viral vectors utilized in this invention may be of various typesincluding hybrid vectors. Vectors may, for instance, be third-generationlentiviral vectors which include only a very small fraction of thenative genome (Zufferey et al., 1998). Production of transgene encodingvector(s) may also involve self-inactivating transfer vectors (Zuffereyet al., 1998; Miyoshi et al., 1998) eliminating the production offull-length vector RNA after infection of target cells.

Viral vectors may be utilized which are replication-incompetent due tofailure to express certain viral proteins necessary for replication.However the possibility exists that helper virus may enable therapeuticvirus replication. This likelihood can be reduced by the use ofself-inactivating vectors.

In a preferred embodiment, transgene sequences are driven by a ubiquitinpromoter, U6 promoter, EFl alpha promoter, CMV promoter, regulablepromoters and/or desired cell type specific promoters.

Viral Tropism

In a preferred embodiment, virus derived from the Numb isoform/Numblikeencoding vector(s), therapeutic vector(s) and/or other transgeneicvector(s) of this invention is pseudotyped with vesicular stomatitisvirus envelope glycoprotein to enable concentration of the virus to hightiters and to facilitate infection of CD34+ cells.

Sequence Selection

The use of any sequence with 70% or greater identity (orcomplementarity) to any sequence referred to as a NUMB or Numblikesequence (searchable using the Entrez-Pubmed database) is covered by theinvention if utilized in the manner described in the present invention.

The current invention also relates in part to a genetic vector thatincludes sequences capable of markedly reducing the susceptibility ofmammalian cells to infection by HIV 1 and IIlV-2 viruses (both togetherreferred to herein as HlV).

The current invention discloses the novel combination of syntheticoligonucleotides to reduce the expression of genes critical to theHIV/AIDS disease process.

The desirability of combining synthetic oligonucleotides to effectco-receptor “knock down” with expression of TAR and RRIi decoysecμjences arises from the proposition, expressed herein, that combiningmultiple gene therapy approaches simultaneously targeting 1) HIVinfection, 2) HlV transcription, and 3) HIV replication in individualcells is likely to produce superior therapeutic benefits than any ofthese approaches in isolation.

Therapeutic vector(s) express “‘beneficial sequence(s)” intended torender transfected/contacted or infected cells less capable ofsustaining HIV replication and transcription. The genetic vectorexpressing “beneficial sequence(s)” as well as any virus derived fromsuch genetic vector, are herein termed “therapeutic vector”.

The present invention is directed in part to the genetic modification ofcells susceptible to infection by HIV or capable of propagating HlV.Such cells are herein termed “target cells”.

The present invention provides a composition and method for usingtherapeutic viral vectors to reduce the susceptibility of mature orimmature target cells, leukocytes, blood cells, any stem/progenitorcells, and/or their progeny to infection by HIV.

It follows that the present invention also provides a composition andmethod for using therapeutic viral vectors to reduce the susceptibilityof reprogrammed cells, induced multipotent cells, induced pluripotentcells, and/or their progeny to infection by HIV.

It is a further objective of this invention to reduce the ability ofmature or immature target cells, stem/progenitor cells, (includingreprogrammed cells, induced multipotent cells, induced pluripotentcells) and/or their progeny to sustain immunodeficiency virusreplication and transcription.

It is another objective of this invention to achieve efficient,long-term expression of the therapeutic sequences in mature or immaturetarget cells, other quiescent cells, stem/progenitor cells, and/or theirprogeny.

In one aspect, this invention provides a method for preventing ortreating HIV infection. The method involves transplanting stem cellstransfected/contacted with therapeutic vector(s) or sequence(s), intopatients with HIV infection.

Beneficial sequence(s) may be ones that reduce the ability of HIV toinfect a cell, transcribe viral DNA, or replicate within an infectedcell, or which enhances the ability of a cell to neutralize HIVinfection.

In certain embodiments, the beneficial sequence(s) represent syntheticoligonucleotide(s) which interfere with HIV entry, including siRNA,shRNA, antisense RNA or miRNA directed against any of the HIVco-receptors (including, but not limited to, CXCR4, CCR5, CCR2b, CCR3,and CCR1).

In a preferred embodiment, the therapeutic vector(s) includes syntheticoligonucleotides targeting one or more HIV co-receptors including CXCR4,CCR5, CCR1, CCR2, CCR3, CXCR6 and/or BOB.

In another preferred embodiment the therapeutic vector(s) includessynthetic oligonucleotides targeting the major HIV co-receptors CXCR4and CCR5

In a further preferred embodiment the therapeutic vector(s) includessynthetic oligonucleotides targeting one or more HIV enzymes such as HIVreverse transcriptase, integrase and protease.

Appropriate sequences for the synthetic oligonucleotides are those 1)predictable by computer algorithms to be effective in reducing targetedsequences, and 2) capable of successfully reduce the amount of targetedenzyme by >70% in standard quantitative RNA assays and in assays ofenzymatic activity or to a lesser but therapeutic degree.

The phrase “targeted sequence” indicates that a particular sequence hasa nucleotide base sequence that has at least 70% identity to a viralgenomic nucleotide sequence or its complement (e.g., is the same as orcomplementary to such viral genomic sequence), or is a corresponding RNAsequence. In particular embodiments of the present invention, the termindicates that the sequence is at least 70% identical to a viral genomicsequence of the particular virus against which the oligonucleotide isdirected, or to its complementary sequence.

Any of the various types of synthetic oligonucleotides may be expressedvia therapeutic vector transfection, and the current invention isdirected to all possible combinations of such oligonucleotides.

In a preferred embodiment, the synthetic oligonucleotide sequences aredriven by target cell, specific promoter(s).

In another preferred embodiment, the synthetic oligonucleotide sequencesare driven by U6 promoter(s).

Synthetic oligonucleotides, by the same token, may be included in thesame therapeutic vector(s) with decoy RNA.

Decoy RNA

Decoy RNA are sequences of RNA that are effective at binding to certainproteins and inhibiting their function.

In a preferred embodiment, the therapeutic vector(s) comprise(s)multiple decoy RNA sequences.

In a further embodiment the decoy RNA sequences are flanked by sequencesthat provide for stability of the decoy sequence.

In another preferred embodiment the decoy RNA sequences are RRE and/orTAR decoy sequences.

In a preferred embodiment, the RRE and TAR decoy sequences are HIV-2derived TAR and RRE sequences.

In another preferred embodiment the decoy sequences also include Psielement decoy sequences.

In a preferred embodiment, the decoy sequences are each driven by a U6promoter.

In another preferred embodiment, the decoy sequences are driven bytarget-cell specific promoters.

In a preferred embodiment, the therapeutic vector targets multiplestages of the HIV life cycle by encoding synthetic nucleotidesequence(s) in combination with HIV-2 TAR and/or RRE decoy sequences.

In another preferred embodiment, the vector includes miRNAoligonucleotide sequences.

In another preferred embodiment, the vector includes shRNAoligonucleotide sequences.

In another preferred embodiment, the vector includes si RNAoligonucleotide sequences.

In another preferred embodiment, the vector includes RNAioligonucleotide sequences.

In another preferred embodiment, the vector includes ribozyme sequences.

In another preferred embodiment, the vector includes a combination ofsynthetic oligonucleotide classes.

In a further embodiment, the synthetic nucleotide sequences target HIVco-receptors such as CCR5, CXCR4, etc.

In a further embodiment, the synthetic nucleotide sequences target HIVenzymes such as integrase, protease, reverse transcriptase, TAT, etc.

In a further embodiment, the ribozyme sequences target HIV co-receptorssuch as CCR5, CXCR4, etc, or HIV enzymes such as integrase, protease,reverse transcriptase, TAT, etc.

In a preferred embodiment, virus is generated using the therapeuticvector(s) and the virus is pseudotyped.

In a preferred embodiment, virus is generated using the therapeuticvector(s) and the virus is not pseudotyped and the virus shows nativeHIV tropism.

In a preferred embodiment, the therapeutic vector(s) is a viral vector.

In a preferred embodiment, the therapeutic vector(s) is a lentiviralvector.

In a preferred embodiment, the therapeutic vector(s) is a thirdgeneration lentiviral vector.

In a preferred embodiment, the therapeutic vector(s) includes acombination of synthetic oligonucleotide classes.

In a preferred embodiment, synthetic nucleotide sequence expression isdriven by the EF-I alpha promoter or other target-cell appropriatepromoters.

In a preferred embodiment, synthetic nucleotide sequence expression isdriven by the 1)6 promoter or other target-cell appropriate promoters.

In a preferred embodiment, synthetic nucleotide sequence expression isdriven by a combination of EF-I alpha and U6, and/or other target-cellappropriate promoters.

In a preferred embodiment, HF-I alpha drives miRNA expression while theU6 promoter drives RNA decoy expression.

In a preferred embodiment, HF-I alpha drives siRNA sequence expressionwhile the U6 promoter drives RNA decoy expression.

In a preferred embodiment, EF-I alpha drives shRNA sequence expressionwhile the U 6 promoter drives RNA decoy expression.

In a preferred embodiment, the therapeutic vector(s) includes multiplemiRNA sequences directed against CXCR4, multiple miRNA sequencesdirected against CCR5, an 111V-2 RRE decoy sequence and an HIV-2 TARdecoy sequence, and the vector is a viral vector.

In a preferred embodiment, treatment involving the therapeutic vector(s)is combined with other modes of antiretroviral therapy includingpharmacological therapies.

Antiretroviral therapies appropriate for combination with thetherapeutic vector(s) are those that have additive or synergisticeffects in combination with the therapeutic vector.

Cells targeted for gene therapy in HIV may include, but are notnecessarily be limited to mature peripheral blood T lymphocytes,monocytes, tissue macrophages, T cell progenitors, macrophage-monocyteprogenitor cells, and/or multipotent hematopoietic stem cells, such asthose found in umbilical cord blood, peripheral blood, and occupyingbone marrow spaces.

The present invention also relates to transfection of CD4+ T cells,macrophages, T cell progenitors, macrophage-monocyte progenitors, CD 34+stem/progenitor cells and/or any other quiescent cell, dividing cell,stem cell or progenitor cell capable of differentiation in vitro or invivo into HIV target cells, CD4+ T cells, macrophages, T cellprogenitors, macrophage-monocyte progenitors, and/or CD 34+stem/progenitor cells. Transfected cells, therefore, can be endogenouscells in situ, or exogenous cells derived from other body regions oreven other individual donors. Cells selected for this purpose are hereintermed “selected cells”.

By the same token, self-renewing, multipotent and/or pluripotent stemcells (including reprogrammed and induced pluripotent cells) representanother logical target for HIV gene therapy, and their use isspecifically covered by the present invention.

In one embodiment of this process, selected cells (e.g. hematopoieticstem cells, skin stem cells, umbilical cord cells, primordial germ cells(PGCs), spermatogonia, any accessible somatic cell, etc.) are 1)propagated in culture using one or more cytokines such as steel factor,leukemia inhibitory factor (LIF), cardiotropic 1, IL-11, IL-6, IL-6 R,GP-130, CNTF, IGF-I, bFGF, and/or oncostatin-M and 2)transfected/contacted with the therapeutic vector(s) or beneficialsequence(s) prior to differentiation using any methods known to the art,such as those described in U.S. Pat. No. 5,677,139 herein incorporatedby reference, or by methods analogous to U.S. Pat. No. 5,677,139 withrespect to other target cells.

In separate embodiments, it may be desirable to perform the varioussteps prior to transfection.

In separate embodiments, for the purpose of generating pluripotent stemcell populations, it may be desirable to perform only the incubationsteps above.

Appropriate concentrations of LlF and steel factor for stem/progenitorcell propagation/proliferation as well as other cell culture conditionshave been described previously (e.g. U.S. Pat. Nos. 6,432,711 and5,453,357 herein incorporated by reference). Other appropriate protocolsand reference cytokine concentrations have been taught by Koshimizu etal., 1996; Keller et al., 1996; Piquet-Pellorce, 1994; Rose et al.,1994; Park and Han, 2000; Guan et al., 2006; Dykstra et al., 2006).

The population of target cells may include somatic cells, stem cells andprogenitor cells. Stem cells may be derived from existing cell lines orisolated from stored, banked, or cryopreserved sources. Typical sourcesof stem cells include marrow, peripheral blood, placental blood,amniotic fluid, umbilical cord blood, adipose tissue, non-human embryos,etc.

Somatic cells, especially circulating leukocytes and othernon-progenitor/stem cells may likewise be subjected to the same cultureconditions as described above for stem/progenitor cells effective thatthey acquire stem/progenitor cell properties as a result.

The invention also discloses the production (e.g. US Patent Application20030099621) of target cells from stem/progenitor cells that may be maderelatively resistant to HIV infection and/or HIV replication.

It is understood, however, that any method of differentiating previouslypropagated stem/progenitor/leukocyte cells into the desired target cellsmay be employed within the scope of the invention so long as functionaltarget cells relatively resistant to HIV infection and/or HIVreplication/and/or HIV transcription are produced.

In a preferred embodiment, the therapeutic viral vector is packaged withone or more envelope proteins from native HIV viruses conferring uponthe therapeutic virus the capacity to infect any cell that native HIVstrains are capable of infecting.

Cells selected for use in this invention will be in some instancesaccessible (e.g. umbilical cord stem cells, bone marrow stem cells,spermatogonia and primordial germ cells of the testis, stem cellsisolated from amniotic fluid, stem cells isolated from the skin, etc.).Such cells can be isolated from the tissues in which they reside by anymeans known to the art.

Other selected cells may comprise reprogrammed cells, inducedmultipotent cells, induced pluripotent cells, etc.

In accordance with an aspect of the present invention, there is provideda method of producing a desired cell line, cell type, or cell class fromthe selected cells. Generally, the method comprises culturing theselected cells and/or their progeny under conditions which promotegrowth of the selected cells at an optimal growth rate. The resultingcell population is then cultured under conditions which promote cellgrowth at a rate which is typically less than the optimal rate, and inthe presence of an agent promoting differentiation of the cells into thedesired cell line, cell type, or cell class (e.g. CD4+ T cells).

The present invention also discloses the propagation of the selectedcells and/or their progeny in culture, before or after transfection withthe therapeutic vector, by any means known to the art (e.g. US PatentApplication 20060099177). Such methods also include incubation with LIF,steel factor, 11-6, IL-7, oncostatin-M and/or cardiotropic 1 and othergrowth enhancing cytokines, etc.

The present invention further discloses the directed differentiation ofcells transfected/contacted with the therapeutic vector(s) into desiredcell types by further incubation in media containing the appropriatecytokines and growth factors such as colony stimulating factors such asM-CSF (CSF-I), GM-CSF, IL-7, any cytokine promoting CD4+ T celldifferentiation, etc.

Transfection

Genetic modification of selected cells and target cells, whether they beexogenous cells or endogenous cells can be performed according to anypublished or unpublished method known to the art (e.g. U.S. Pat. Nos.6,432,711, 5,593,875, U.S. 5,783,566, U.S. Pat. Nos. 5,928,944,5,910,488, 5,824,547, etc.) or by other generally accepted means.Suitable methods for transforming host cells can be found in Sambrook etal.

(Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring HarborLaboratory press (1989)), and other laboratory textbooks.

Successfully transfected/contacted cells can be identified by selectionprotocols involving markers such as antibiotic resistance genes inaddition to RNA expression assays and morphological analyses. Clonesfrom successfully transfected/contacted cells, expressing theappropriate exogenous DNA at appropriate levels, can be preserved ascell lines by cryopreservation (utilizing any appropriate method ofcryopreservation known to the art).

Selectable markers (e.g., antibiotics resistance genes) may includethose which confer resistance to drugs, such as G418, hygromycin,ampicillin and blasticidin, etc. Cells containing the gene of interestcan be identified by drug selection where cells that have incorporatedthe selectable marker gene survive, and others die.

A theoretical basis for the embodiments of the invention is describedherein, however, this discussion is not in any way to be considered asbinding or limiting on the present invention. Those of skill in the artwill understand that the various embodiments of the invention may bepracticed regardless of the model used to describe the theoreticalunderpinnings of the invention.

The invention will now be described and illustrated with respect to thefollowing examples; however, the scope of the present invention is notintended to be limited thereby.

Example 1: Construction of the Transgenic Vectors Suitable for Use inthe Present Invention

Suitable EGFP-Numb and HGFP-Numblike, and EGFP-X lentiviral vectors(where X is any transgene described in the present invention) can beproduced by cloning into an appropriate viral vector (e.g. the two-geneI Il V-EGFP-HSA vector (Reiser et al., 2000)). Adapter primers can beselected for PCR amplification of Numblike and Numb isoform cDNAs andcloning into a genetic vector. In preparation for cloning, the genevector is digested with enzymes. Subsequently, the cDNA for eachtransgene is inserted into the nef coding region previously occupied bythe I ISA cDNA. EGFP (enhanced green fluorescent protein) and a cellpopulation-appropriate promoter (e.g. CMV ie or EFl alpha) having beenpreviously inserted into the viral coding region. Genetic constructs mayinclude a vector backbone, and a transactivator which regulates apromoter operably linked to heterologous nucleic acid sequences.

Examples of retroviral vectors which may be employed include, but arenot limited to, those derived from Moloney Murine Leukemia Virus,Moloney Murine Sarcoma Virus, and Rous Sarcoma Virus, FIV, and HIV.Appropriate expression vectors are those that may be employed fortransfecting DNA or RNA into eukaryotic cells. Such vectors include, butare not limited to, prokaryotic vectors such as, for example, bacterialvectors; eukaryotic vectors, such as, for example, yeast vectors andfungal vectors; and viral vectors, such as, but not limited to,lentiviral vectors, adenoviral vectors, adeno-associated viral vectors,and retroviral vectors.

The replication incompetent pcDNA 6.2/EmGFP-Bsd/V5-DEST vector is anexample of an appropriate expression vector (Invitrogen) and allowsexpression of synthetic oligonucleotides (e.g. miRNAs) transferred fromthe pcDNA 6.2 GW/miR vector that have the capacity to cleave targetedsequences. These vectors include flanking and loop sequences fromendogenous miRNA to direct the excision of the engineered miRNA from alonger Pol II transcript (pre-miRNA).

Combining multiple miRNA sequences directed against specific endogenousRNA species increases the likelihood of success in reducing targetsequence expression. miRNA sequences may be operably linked to regulableor tissue specific promoters.

By utilizing lentiviral vectors for gene expression, the resultingNumb/Numblike encoding vector(s) and/or other transgenic vector(s) ofthis invention, becomes capable of stably transducing both dividing andnon-dividing cell types.

In a preferred embodiment, the resulting Numb/Numblike encodingvector(s), and/or other transgenic vector(s) of this invention containmultiple synthetic oligonucleotide sequences driven by one or morepromoters so as to reduce expression of specific numb isoforms and/ornumblike.

Example 2

Another example of a suitable vector is a retroviral vector.Retroviruses are RNA viruses that contain an RNA genome. The gag, pol,and env genes are flanked by long terminal repeat (LTR) sequences. The5′ and 3′ LTR sequences promote transcription and polyadenylation ofmRNA's.

The retroviral vector may provide a regulable transactivating element,an internal ribosome reentry site (IRHS), a selection marker, and atarget heterologous gene operated by a regulable promoter.

Alternatively, multiple sequences may be expressed under the control ofmultiple promoters. Finally, the retroviral vector may containcis-acting sequences necessary for reverse transcription andintegration. Upon infection, the RNA is reverse transcribed to DNA thatintegrates efficiently into the host genome. The recombinant retrovirusof this invention is genetically modified in such a way that some of theretroviral, infectious genes of the native virus have been removed andin certain instances replaced instead with a target nucleic acidsequence for genetic modification of the cell. The sequences may beexogenous DNA or RNA, in its natural or altered form.

Example 3: Example Methods for Generation of Numb/Numblike EncodingVector(s), and/or Other Transgenic Vector(s) of this Invention

The methods for generation of the resulting Numb/Numblike encodingvector(s), and/or other transgenic vector(s) of this invention includethose taught in Invitrogen's Viral Power Lentiviral Expression SystemsManual, 2007. Briefly, the EmGFP-bsd cassette is cloned as a PmIl-BIpIfragment into the pLenti6/R4R2/V5-DEST vector, while the mill-long(PRR+) numb isoform or miR-short numb isoform/numblike cassettes aresimultaneously transferred by BP reaction into pDONR221. Then theregulable promoter(s) and miR-isoform cassettes are Multisite LR crossedinto the modified pLenti6/EmGFP-bsd/R4R2-DES Tvector.

Multiple vectors can be generated in this manner comprising differentcombinations of synthetic oligonucleotides and transgene cassettes.

pLenti6/R4R2/V5-DEST vector sequence: (SEQ ID NO: 1)aatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtatttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgltgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggltcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgatgtcgacgttaacgctagtgatatcaactttgtatagaaaagttgaacgagaaacgtaaaatgatataaatatcaatatattaaattagattttgcataaaaaacagactacataatactgtaaaacacaacatatccagtcactatggcggccgcattaggcaccccaggctttacactttatgcttccggctcgtataatgtgtggattttgagttaggatccgtcgagattttcaggagctaaggaagctaaaatggagaaaaaaatcaclggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgatlcaggttcatcatgccgtttgtgatggcttccatgtcggcagaatgcttaatgaattacaacagtactgcgatgagtggcagggcggggcgtaaagatctggatccggcttactaaaagccagataacagtatgcgtatttgcgcgctgattttlgcggtataagaatatatactgatatgtatacccgaagtatgtcaaaaagaggtatgctatgaagcagcgtattacagtgacagttgacagcgacagctatcagttgctcaaggcatatatgatgtcaatatctccggtctggtaagcacaaccatgcagaatgaagcccgtcgtctgcgtgccgaacgctggaaagcggaaaatcaggaagggatggctgaggtcgcccggtttattgaaatgaacggctcttttgctgacgagaacagggactggtgaaatgcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtgatattattgacacgcccgggcgacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaactttacccggtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgccggtctccgttatcggggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatataaatgtcaggctccgttatacacagccagtctgcaggtcgaccatagtgactggatatgttgtgttttacagtattatgtagtctgttttttatgcaaaatctaatttaatatattgatatttatatcattttacgtttctcgttcagctttcttgtacaaagtggttgatatccagcacagtggcggccgctcgagtctagagggcccgcggttcgaaggtaagcctatccctaaccctctcctcggtctcgattctacgcgtaccggttagtaatgagtttggaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttaggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcagcacgtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaciggtgaggaactaaaccatggccaagcctttgtctcaagaagaatccaccctcattgaaagagcaacggctacaatcaacagcatccccatctctgaagactacagcgtcgccagcgcagctctctctagcgacggccgcatcttcactggtgtcaatgtatatcattttactgggggaccttgtgcagaactcgtggtgctgggcactgctgctgctgcggcagctggcaacctgacttgtatcgtcgcgatcggaaatgagaacaggggcatcttgagcccctgcggacggtgccgacaggtgcttctcgatctgcatcctgggatcaaagccatagtgaaggacagtgatggacagccgacggcagttgggattcgtgaattgctgccctctggttatgtgtgggagggctaagcacaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttltagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaaUtcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagtlctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagctt

Example 4: Additional Methods for Generation of Therapeutic Vector(s)

“Packaging cell lines” derived from human and/or animal fibroblast celllines result from transfecting or infecting normal cell lines with viralgag, pol, and env structural genes. On the other hand, packaging celllines produce RNA devoid of the psi sequence, so that the viralparticles produced from packaging cell do not contain the gag, pol, orenv genes. Once the therapeutic vector's DNA containing the psi sequence(along with the therapeutic gene) is introduced into the packaging cell,by means of transfection or infection, the packaging cell may producevirions capable of transmitting the therapeutic RNA to the final targetcell (e.g. a CD4+ cell).

The “infective range” of the therapeutic vector(s) is determined by thepackaging cell line. A number of packaging cell lines are available forproduction of virus suitable for infecting a broad range of human celltypes. These packaging cell lines are nevertheless generally capable ofencapsidating viral vectors derived from viruses that in nature usuallyinfect different animal species. For example, vectors derived from SIVor MMLV can be packaged by GP 120 encapsidating cell lines.

An example protocol for producing a therapeutic viral supernatant isprovided as follows:

1. Twenty micrograms of retrovirus vector are mixed with 2-3 microgramsof viral DNA containing the selectable marker gene (e.g. antibioticresistance gene) by gentle tapping in 0.8-1 milliliter of Ilepesbuffered saline (pH=7.05) in a 1.5 ml plastic tube.

2. Seventy microliters of 2M CaCl are added to the mixture by repeatedgentle tapping.

3. When a blue precipitate first begins to appear within the tube, theproduct should be gently applied to a 30% confluent layer of packagingcells (from any number of commercial vendors). The DNA mixture should beapplied only after first removing the medium from the packaging cells.

4. The packaging cells are set to incubate for 20-30 minutes at roomtemperature (25 degrees Celsius) before transferring them back to anincubator at 36-38 degrees Celsius for 3.5 hours.

5. Add 3.5-4 milliliters of Hepes buffered saline containing 15%glycerol for 3 minutes then wash cell with Dulbecco's Modified Eagle'sMedium (DMEM)+! 0% FBS×2.

6. Add back DMKM+10% FBS, and incubate cells for 20 hours at 37 degreesCelsius.

7. Remove and filter medium containing therapeutic viral particles.

Excess viral supernatant is immediately stored or concentrated andstored at −80 degrees Celsius). Supernatant may stored with 5-8micrograms of polybrene to increase the efficiency of target cellinfection. Otherwise polybrene may be excluded or added just beforeinfection.

8. Stable producer lines can be established by splitting packaging celllines 1 to 20, or 1 to 40 and subsequently incubating these cells for upto 10 days (changing medium every three days) in medium containingselective drugs (e.g. certain antibiotics corresponding to transfectedresistance genes).

9. After 10 days isolated colonies are picked, grown-up aliquoted andfrozen for storage.

Assay of Retrovirus Infectivity/Titration is achieved by application ofa defined volume of viral supernatant to a layer of confluent “test”cells such as NIII 3T3 cells plated at 20% confluence. After 2-3 celldivision times (24-36 hours for NIH 3T3 cells) colonies of “test” cellsincubated at 37 degrees in antibiotic-containing medium are counted. Thesupernatant's titer are estimated from these colony counts by thefollowing formula:

Colony Forming Units/ml=colonies identified×0.5(split factor)/volume ofvirus(ml)

The accuracy of this estimate is increased by testing large volumes ofsupernatant over many plates of “test” cells.

Application of the therapeutic viral supernatant to target cells may beaccomplished by various means appropriate to the clinical situation.

Example 5: Growth Medium for Selected Cells

Selected cells can be expanded/grown in Dulbecco's modified MinimalEssential Medium (DMEM) supplemented with glutamine,beta.-mercaptoethanol, 10% (by volume) horse serum, and humanrecombinant Leukemia Inhibitory Factor (LIF). LIF replaces the need formaintaining selected cells on feeder layers of cells, (which may also beemployed) and is essential for maintaining selected cells in anundifferentiated, multipotent, or pluripotent state, such cells can bemaintained in Dulbecco's modified Minimal Essential Medium (DMEM)supplemented with glutamine, beta.-mercaptoethanol, 10% (by volume)horse serum, and human recombinant Leukemia Inhibitory Factor (LIF). TheLIF replaces the need for maintaining cells on feeder layers of cells,(which may also be employed) and is essential for maintaining cells inan undifferentiated state (per U.S. Pat. No. 6,432,711).

In order to initiate the differentiation of the selected cells intoneuronal cells, the cells are trypsinized and washed free of LIF, andplaced in DMEM supplemented with 10% fetal bovine serum (FBS). Afterresuspension in DMEM and 10% FBS, IX105 cells are plated in 5 ml DMEM,10% FBS, 0.5 microM retinoic acid in a 60 mm Fisher bacteriologicalgrade Petri dishes, where the cells are expected to form smallaggregates. Aggregation aids in proper cell differentiation. Highefficiency transfection with appropriate neuronal transcription factorscan occur before or after plating in DMEM, FBS, and retinoic acid. (SeeU.S. Pat. Nos. 6,432,711 and 5,453,357 for additional details).

Example 6: Hla Matching

Selected cells (e.g. umbilical cord blood or cells from any othersuitable source and/or their progeny), can be screened,genetically-modified (optional), expanded, and induced to begindifferentiating into the desired cell type(s) (optional). The cells arethen transplanted according to standard stem cell transplantationprotocols. In certain instances, cells may be transplanted into patientswithout HLA matching.

Example 7

In some rare instance, it may be appropriate to introduce transgeneencoding vectors into patients in order to stimulate or inhibit cellulardivision or cellular differentiation, in vivo.

Example 8: Genetic Modification of Selected Cells

In vitro genetic modification of exogenous cells or patient's endogenouscells can be performed according to any published or unpublished methodknown to the art (e.g. U.S. Pat. Nos. 6,432,711, 5,593,875, 5,783,566,5,928,944, 5,910,488, 5,824,547, etc.) or by other generally acceptedmeans. Suitable methods for transforming host cells can be found inSambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition,Cold Spring Harbor Laboratory press (1989)), and other laboratorytextbooks.

Successfully transfected/contacted cells are identified by selectionprotocols involving markers such as antibiotic resistance genes inaddition to RNA expression assays and morphological analyses. Clonesfrom successfully transfected/contacted cells, expressing theappropriate exogenous DNA at appropriate levels, can be preserved ascell lines by cryopreservation (utilizing any appropriate method ofcryopreservation known to the art).

Selectable markers (e.g., antibiotics resistance genes) may includethose conferring resistance to drugs, such as G418, hygromycin andmethotrexate. Cells containing the gene of interest can be identified bydrug selection where cells that have incorporated the selectable markergene survive, and others die.

The current invention discloses the selection of genetically-modifiedcells as “selected cells” of the invention. The term geneticmodification refers to alteration of the cellular genotype byintroducing natural or synthetic nucleic acids into selected cellsand/or their progeny or immortalized cell lines and/or their progeny byany means known to the art. Alternatively culture conditions that inducepermanent changes in gene expression patterns are considered herein torepresent genetic modification. Modification of stem cells, whether theybe derived from the host brain, endogenous donor sources, exogenousdonor sources, or cell lines, represents a feasible approach to thetreatment of certain human diseases, especially those of the humannervous system.

Genetic modifications covered by this disclosure include, but are notlimited to: genetic modifications performed in vivo; modifications thatalter the activity or amount of metabolic enzymes expressed byendogenous or exogenous selected cells and/or their progeny;modifications which alter the activity, amount, or antigenicity ofcellular proteins; modifications which alter the activity or amount ofproteins involved in signal transduction pathways; modifications whichalter HLA type; modifications which alter cellular differentiation;modifications which alter neoplastic potential; modifications whichalter cellular differentiation; modifications which alter the amount oractivity of structural proteins; modifications which alter the amount oractivity of membrane associated proteins (structural or enzymatic);modifications which alter the activity or amount of proteins involved inDNA repair and chromosome maintenance; modifications which alter theactivity or amount of proteins involved in cellular transport;modifications which alter the activity or amount of enzymes;modifications which alter the activity or amount of proteins involved insynapse formation and maintenance; modifications which alter theactivity or amount of proteins involved in neurite outgrowth or axonoutgrowth and formation; modifications altering the amount or activityof antioxidant producing enzymes within the cell; modifications whichlead to altered post-translational modification of cellular proteins;modifications which alter the activity or amount of proteins involved inother aspects of cellular repair, and alterations which increase thelifespan of the cell (such as production of telomerase). Such proteinsas those mentioned above may be encoded for by DNA or RNA derived fromthe human genome or other animal, plant, viral, or bacterial genomes.This invention also covers sequences designed de novo.

In addition, this invention relates to the in situ, genetic modificationof selected cells and/or their progeny cells for the treatment ofdisease. Endogenous stem cells may be modified in situ by directinjection or application of DNA or RNA vectors, including viruses,retroviruses, liposomes, etc, into the substance of the tissue or intothe appropriate portion of the ventricular system of the brain. Since1992, we have modified thousands of stem/progenitor cells and manythousand progeny cells in this manner. Our data shows that this mannerof modifying progenitor cells results in a tremendous variety ofmodified cell types throughout the nervous system, and has neverresulted in adverse effects.

Example 9: Introduction of Genetic Vectors into the Host

In a preferred embodiment, endogenous cells are transfected/contactedwith vectors such as those described herein in vivo by introduction ofthe therapeutic vector(s) into the host blood, tissues, nervous system,bone marrow, etc. The greatest benefit may be achieved by modifying alarge number of endogenous target cells. This may be accomplished byusing an appropriately-sized, catheter-like device, or needle to injectthe therapeutic vector(s) into the venous or arterial circulation, intoa specific tissue, such as muscle tissue, or into the nervous system. Ina preferred embodiment, the virus is pseudotyped with VSV-G envelopeglycoprotein and native HIV-I env proteins.

Example 10: Injection into the Nervous System

Transplantation of selected cells (from either the growth ordifferentiation media) into the fetal nervous system or geneticmodification of endogenous fetal cells utilizing genetic vectors may beaccomplished in the following manner Under sterile conditions, theuterus and fetuses are visualized by ultrasound or other radiologicalguidance. Alternatively the uterus may be exposed surgically in order tofacilitate direct identification of fetal skull landmarks. Selectedcells can then be introduced by injection (using an appropriately-sizedcatheter or needle) into the ventricular system, germinal zone(s), orinto the substance of the nervous system. Injections may be performed incertain instances, through the mother's abdominal wall, the uterine walland fetal membranes into the fetus. The accuracy of the injection ismonitored by direct observation, ultrasound, contrast, or radiologicalisotope based methods, or by any other means of radiological guidanceknown to the art.

Under appropriate sterile conditions, direct identification of fetalskull landmarks is accomplished visually as well as by physicalinspection and palpation coupled with stereotaxic and radiologicguidance. Following cell culture, appropriate amounts of the selected ordifferentiating cells can then be introduced by injection or other meansinto the ventricular system, germinal zones, or into the substance ofthe nervous system. The accuracy of the injection may be monitored bydirect observation, ultrasound, or other radiological guidance.

In certain, neurological diseases of the adult nervous system, such asHuntington's disease and Parkinson's disease, cells of a specificportion of the brain are selectively affected. In the case ofParkinson's disease, it is the dopaminergic cells of the substantianigra. In such regionally-specific diseases affecting adults, localizedtransplantation of cells may be accomplished by radiologically-guidedtransplantation of differentiating cells under sterile conditions.Radiologic guidance may include the use of CT and/or MRI, and may takeadvantage contrast or isotope based techniques to monitor injectedmaterials.

In certain neurologic diseases, such as some metabolic storagedisorders, cells are affected across diverse regions of the nervoussystem, and the greatest benefit may be achieved bygenetically-modifying endogenous cells or introducing selected cells ofthe present invention (either from the growth culture media or thedifferentiating medium) into the tissue in large numbers in a diffusemanner. In the nervous system, these diseases may be best approached byintraventricular injections (using an appropriately-sized, catheter-likedevice, or needle) (especially at early stages of development) whichallows diffuse endogenous cell modification or diffuse engraflment ofselected cells isolated from the growth and/or differentiation media.Nevertheless, injection of the cells into the circulatory system for thesame purpose is also covered. However, with regard to any disorderaffecting multiple organs or the body diffusely (e.g. lysosomal storagedisorders, hemoglobinpathies, muscular dystrophy), the cells isolatedfrom the growth and/or differentiation media may also be preferentiallyintroduced directly into the circulation and/or visceral organs, such asthe liver, kidney, gut, spleen, adrenal glands, pancreas, lungs, andthymus using endoscopic guidance and any appropriately-sized,catheter-like device, allowing diffuse engraftment of the cellsthroughout the body, as well as specific introduction and infiltrationof the cells into the selected organs.

Example 11: Delivery of Cells by Injection in to the Circulatory Streamand Organs

Diseases of one organ system may be treatable with genetically modifiedcells from a separate organ system. Also, in some instances, it maybecome apparent that the selected cells may integrate and differentiateon their own, in vivo, in sufficient numbers if they are injected intoblood stream either arterial, venous or hepatic, after culturing in thegrowth and/or differentiation media. This approach is covered by thepresent invention. The treatment of diffuse muscle (e.g. musculardystrophies), organ, tissue, or blood disorders (e.g. HereditarySpherocytosis, Sickle cell anemia, other hemoglobinopathies, etc,) may,for instance, involve the injection of cells isolated from the growthmedia or differentiating media into the patient, especially thepatient's circulation. This approach is also believed to ameliorateischemic injuries such as myocardial infarction, stroke, etc., as wellas traumatic injuries to brain and other tissues. Injection of suchcells produced by the current invention, directly into the circulation,by needle or catheter, so that the cells are enabled to “home” to thebone marrow, muscle, kidneys, lungs, and/or any other other organsystem, as well as injection directly into the bone marrow space issuitable for the practice of the present invention. Likewise injectionof the cells directly into a lesion site with or without radiologic,ultrasonic or fluoroscopic guidance is also suitable for the practice ofthe present invention.

Methods of isolating selected cells useful in the present inventioninclude those described by Zhao et al., 2006.

In a preferred embodiment, genetic vectors encoding numblike and/or numbisoforms comprise regulable promoters operably linked to the Numb ornumblike transgenes.

In another preferred embodiment, the mode of transfection may beselected from those modes of transfection/contacting that provide fortransient rather than permanent expression of the numblike and numbisoforms.

Example 12

Example Genetic Modifications It is believed that hundreds of diseasesand clinical conditions are able to be treated and/or ameliorated by themethods of the present invention including, but in no way limited toCanavan's disease (ASP); Tay-Sach's disease (HEXA); Lesch-Nyhan syndrome(HRPT); Huntington's disease(HTT); Sly syndrome; type A and type BNiemann Pick disease;

Sandhoff s disease (HEXB); Fabry's disease (GLA); type C Niemann-Pickdisease(NPC1); Gaucher's disease (GBA); Parkinson's disease(PARK2,etc.); Von Hippel Lindau's disease, Sickle cell anemia (HBB) and otherthalassemias as well as similar diseases. These transgenes may representthe coding region or portions of the coding region of the normal genes.

It is to be understood, however, that the scope of the present inventionis not to be limited to the specific embodiments and examples describedabove. The invention may be practiced other than as particularlydescribed and still be within the scope of the accompanying claims.

Example 13

An example sequence for a vector capable of rendering cells pluripotentand expressing a long Numb isoform, Oct-4, Sox-2, and EmGFP nucleic acidsequences under the control of tetracycline-sensitive promoters is:

(SEQ ID NO: 2)aatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtatttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactciggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgatgtcgacgttaacgctagtgatatcaactttgtatagaaaagttgaacgagaaacgtaaaatgatataaatatcaatatattaaattagattttgcataaaaaacagactacataatactgtaaaacacaacatatccagtcactatgggacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggaaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctccctatcagtgatagagatctccctatcagtgatagagatcgtcgacgagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctccggactctagcgtttaaacttaagcttaccatgccttcgcaagccctcatttcaccaggcccccggcttggggcgccttccttccccatggcgggacacctggcttcggatttcgccttctcgccccctccaggtggtggaggtgatgggccaggggggccggagccgggctgggttgatcctcggacctggctaagcttccaaggccctcctggagggccaggaatcgggccgggggttgggccaggctctgaggtgtgggggattcccccatgccccccgccgtatgagttctgtggggggatggcgtactgtgggccccaggttggagtggggctagtgccccaaggcggcttggagacctctcagcctgagggcgaagcaggagtcggggtggagagcaactccgatggggcctccccggagccctgcaccgtcacccctggtgccgtgaagctggagaaggagaagctggagcaaaacccggaggagtcccaggacatcaaagctctgcagaaagaactcgagcaatttgccaagctcctgaagcagaagaggatcaccctgggatatacacaggccgatgtggggctcaccctgggggttctatttgggaaggtattcagccaaacgaccatctgccgctttgaggctctgcagcttagcttcaagaacatgtgtaagctgcggcccttgctgcagaagtgggtggaggaagctgacaacaatgaaaatcttcaggagatatgcaaagcagaaaccctcgtgcaggcccgaaagagaaagcgaaccagtatcgagaaccgagtgagaggcaacctggagaatttgttcctgcagtgcccgaaacccacactgcagcagatcagccacatcgcccagcagcttgggctcgagaaggatgtggtccgagtgtggttctgtaaccggcgccagaagggcaagcgatcaagcagcgactatgcacaacgagaggattttgaggctgctgggtctcctttctcagggggaccagtgtcctttcctctggccccagggccccattttggtaccccaggctatgggagccctcacttcactgcactgtactcctcggtccctttccctgagggggaagcattccccctgtctccgtcaccactctgggctctcccatgcattcaaactgaggtgcctgcccttctaggaatgggggacagggggaggggaggagctagggaaagaaaacctggagtttgtgccagggtttttgggattaagttcttcattcactaaggaaggaattgggaacacaaagggtgggggcaggggagtttggggcaactggttggagggaaggtgaagttcaatgatgctcttgattttaatcccacatcatgtatcacttttttcttaaataaagaagcctgggacacagtagatagacacacttaaaaaaaaaaacctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgggacggatcgggagatctcccgatcccctatggtgcactctcagtacaatcttgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaa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A schematized map corresponding to the vector sequence above is shown inFIG. 5.

The vector may be constructed fully through de novo gene synthesis, orin part through the cloning of the Numb, Sox and OCT3/4 cDNA sequencesinto the position occupied by LacZ in the Invitrogen pcDNA4tolacZvector. Similarly, the tetR gene is found in the Invitrogen pcDNA6/TRvector. Coding sequences of genes referenced are also appropriate forcloning into the pcDNA41acZ vector.

Alternatively, the tetR gene may be transfected into target cellsseparately utilizing the pcDNA6/TR vector in combination with a vectorcomprising the sequence here minus the tetR gene and its PCMV promoter.

Likewise, multiple vectors may be employed so long as elements similarto the elements included in the above sequence are present. This mayreduce the likelihood of promoter competition. It is to be understoodthat other conditional promoter elements may be substituted for thetetracycline sensitive promoter elements.

Example 14

It is expected that intravenous and other administration of pluripotentstem cells produced according to the methods described herein (or otherpublished methods) one or more times can provide replacement cells tothe body and that such administration may serve to extend the life orimprove the health of the patient suffering age-related senescence.

Example 15: Production of Germ Cells

The current invention covers the derivation of germ cells frommultipotent, pluripotent, and/or self-renewing stem cells producedaccording to the methods described herein (or according to otherpublished methods). The production of such germ cells may be suitablefor treating infertility and producing embryos in vitro (e.g. Ilubner etal., 2003; Kehler et al., 2005; Nayernia et al., 2006a; Nayernia et al.,2006b; Drusenheimer et al., 2007; Moore et al., 2007; etc.)

Example 16: Generation of Transgenic Animals

The present invention covers the generation of transgenic animals. Aswith other pluripotent cells, the pluripotent cells produced by themethods described herein (or other published methods) may be utilized toproduce transgenic animals by any method known to the art.

Example 17: Therapeutic Vector Construction

Examples of retroviral vectors which may be employed include, but arenot limited to, those derived from Moloney Murine Leukemia Virus,Moloney Murine Sarcoma Virus, and Rous Sarcoma Virus, FIV, and HIV.Appropriate expression vectors are that may be employed for transfectingDNA or RNA into eukaryotic cells. Such vectors include, but are notlimited to, prokaryotic vectors such as, for example, bacterial vectors;eukaryotic vectors, such as, for example, yeast vectors and fungalvectors; and viral vectors, such as, but not limited to, lentiviralvectors, adenoviral vectors, adeno-associated viral vectors, andretroviral vectors.

The replication incompetent pcDNA 6.2 GW/miR and pcDNA6.2/EmGFP-Bsd/V5-DEST vectors are examples of an appropriate expressionvectors (Invitrogen) and allow expression of synthetic oligonucleotides(e.g. miRNAs) that have the capacity to cleave targeted sequences. Thesevectors include flanking and loop sequences from endogenous miRNA todirect the excision of the engineered miRNA from a longer Pol IItranscript (pre-miRNA).

Alternatively, inclusion of the HIV psi sequence allows the therapeuticvector to compete with native HIV genome for packaging into viralparticles, also inhibiting HIV transmission.

Combining multiple miRNA sequences directed against a single targetincreases the likelihood of success in reducing target sequenceexpression. miRNA sequences may be operably linked to tissue specificpromoters such as the EF-I alpha promoter, any T cell specific promoter,or macrophage specific promoter to ensure expression in the desired celltypes.

Utilizing Invitrogen's lentiviral destination (DEST) vectors for geneexpression, the resulting therapeutic vector(s) becomes capable ofstably transducing both dividing and non-dividing cell types.

In a preferred embodiment, the therapeutic vector(s) contains multiplesynthetic oligonucleotide sequences driven by one or more promoters soas to reduce expression of CXCR4, CCR5, and/or any other cellularprotein known to act as a co-receptor for HIV infection in target cells.

In one therapeutic vector (constructed in 2006), four miRNA sequencestargeting CXCR4 and CCR5 co-receptors were cloned into the pcDNA 6.2GW/miR vector along with decoy RNA sequences targeting HIV-2 TAR andRRE.

Genetic constructs may include a vector backbone, and a transactivatorwhich regulates a promoter operably linked to heterologous nucleic acidsequences.

Another example of a suitable vector is a retroviral vector.Retroviruses are RNA viruses which contain an RNA genome. The gag, pol,and env genes are flanked by long terminal repeat (LTR) sequences. The5′ and 3′ LTR sequences promote transcription and polyadenylation ofmRNA's.

The retroviral vector may provide a regulable transactivating element,an internal ribosome reentry site (IRES), a selection marker, and atarget heterologous gene operated by a regulable promoter.

Alternatively, multiple sequences may be expressed under the control ofmultiple promoters. Finally, the retroviral vector may containcis-acting sequences necessary for reverse transcription andintegration. Upon infection, the RNA is reverse transcribed to DNA whichintegrates efficiently into the host genome. The recombinant retrovirusof this invention is genetically modified in such a way that some of theretroviral, infectious genes of the native virus are removed and inembodiments replaced instead with a target nucleic acid sequence forgenetic modification of the cell. The sequences may be exogenous DNA orRNA, in its natural or altered form.

Example 18: Example Methods for Generation of the Therapeutic Vector

The methods for generation of the therapeutic vector(s) include thosetaught in Invitrogen's Viral Power Lentiviral Expression Systems Manual(incorporated by reference herein). Briefly, the EmGFP-bsd cassette iscloned as a PmII-BIpI fragment into the pLenti6/R4R2/V5-DEST vector,while the miR-decoy cassette is simultaneously transferred by BPreaction into pDONR221. Then the EFIa promoter and miR-decoy areMultisite LR crossed into the modified pLenti6/KmGFP-bsd/R4R2-DESTvector.

pLenti6/R4R2/V5-DEST vector sequence: (SEQ ID NO: 1)aatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtattlaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctclggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagclagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgatgtcgacgttaacgctagtgatatcaactttgtatagaaaagttgaacgagaaacgtaaaatgatataaatatcaatatattaaattagattttgcataaaaaacagactacataatactgtaaaacacaacatatccagtcactatggcggccgcattaggcaccccaggctttacactttatgcttccggctcgtataatgtgtggattttgagttaggatccgtcgagattttcaggagctaaggaagctaaaatggagaaaaaaatcactggatataccaccgttgatatalcccaatggcatcgtaaagaacaUttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggUcatcatgccgtttgtgatggcttccatgtcggcagaatgcttaatgaattacaacagtactgcgatgagtggcagggcggggcgtaaagatctggatccggcttactaaaagccagataacagtatgcgtatttgcgcgctgatttttgcggtataagaatatatactgatatgtatacccgaagtatgtcaaaaagaggtatgctatgaagcagcgtattacagtgacagttgacagcgacagctatcagttgctcaaggcatatatgatgtcaatatctccggtctggtaagcacaaccatgcagaatgaagcccgtcgtctgcgtgccgaacgctggaaagcggaaaatcaggaagggatggctgaggtcgcccggtttattgaaatgaacggctcttttgctgacgagaacagggactggtgaaatgcagtltaaggttlacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtgatattattgacacgcccgggcgacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaactttacccggtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgccggtctccgttatcggggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatataaatgtcaggctccgttatacacagccagtctgcaggtcgaccatagtgactggatatgttgtgttttacagtattatgtagtctgttttttatgcaaaatctaatttaatatattgatatttatatcattttacgtttctcgttcagctttcttgtacaaagtggttgatatccagcacagtggcggccgctcgagtctagagggcccgcggttcgaaggtaagcctatccctaaccctctcctcggtctcgattctacgcgtaccggttagtaatgagtttggaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttattlatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcagcacgtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagcctttgtctcaagaagaatccaccctcattgaaagagcaacggctacaatcaacagcatccccatctctgaagactacagcgtcgccagcgcagctctctctagcgacggccgcatcttcactggtgtcaatgtatatcattttactgggggaccttgtgcagaactcgtggtgctgggcactgctgctgctgcggcagctggcaacctgacttgtatcgtcgcgatcggaaatgagaacaggggcatcttgagcccctgcggacggtgccgacaggtgcttctcgatctgcatcctgggatcaaagccatagtgaaggacagtgatggacagccgacggcagttgggattcgtgaattgctgccctctggttatgtgtgggagggctaagcacaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctuttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagctt.Example miR-Decoy Cassette Sequence:

(SEQ ID NO: 3)gtcgaccagtggatcctggaggcttgctgaaggctgtatgctgatcgggtgtaaactgagcttggttttggccactgactgaccaagctcattacacccgatcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatcctggaggcttgctgaaggctgtatgctgataccaggcaggataaggccagttttggccactgactgactggccttactgcctggtatcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatcctggaggcttgctgaaggctgtatgctgtgaccaggatgaccaatccatgttttggccactgactgacatggattgcatcctggtcacaggacacaaggcctgttactagcactcacatggaacaaatggcccagatcctggaggcttgctgaaggctgtatgctgatagcttggtccaacctgttagttttggccactgactgactaacaggtgaccaagctatcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctccccagtggaaagacgcgcaggcaaaacgcaccacgtgacggagcgtgaccgcgcgccgagcgcgcgccaaggtcgggcaggaagagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttgggtttatatatcttgtggaaaggacggtgctcgcttcggcagcacgtcgtgctagggttcttgggttttctcgcaacagcaggttctgcaatgggcgcggcgtccctgaccgtgtcggctcagtcccggactttactggccgggatagtgcagcaacagcaacagctgttggacgtggtcaagagacaacaagaactgttgcgactgaccgtctggggaacgaaaaacctccaggcaagagtcactgctatagagaagtacctacaggaccaggcgcggctaaattcatggggatgtctagacctagagcggacttcggtccgctttttccccagtggaaagacgcgcaggcaaaacgcaccacgtgacggagcgtgaccgcgcgccgagcgcgcgccaaggtcgggcaggaagagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttgggtttatatatcttgtggaaaggacggtgctcgcttcggcagcacgtcggtcgctctgcggagaggctggcagattgagccctgggaggttctctccagcactagcaggtagagcctgggtgttccctgctagactctcaccagtgcttggccggcactgggcagacggctccacgcttgcttgcttaaagacctcttaataaagctgctctagacctagagcggacttcggtccgcttttttacgtactcgag.

Example 19: Methods for Propagating/Proliferating Stem/Progenitor CellsIn Vivo

In order to obtain large numbers of target cells that are relativelyresistant to 1) HIV infection and/or 2) HIV replication and/or 3) HIVtranscription, progenitor/stem cells can be grown in Dulbecco's modifiedMinimal Essential Medium (DMEM) supplemented with glutamine,beta.-mercaptoethanol, 10% (by volume) horse serum, and humanrecombinant Leukemia Inhibitory Factor (LIF). The LIF replaces the needfor maintaining progenitor/stem cells on feeder layers of cells, (whichmay also be employed) and is essential for maintaining progenitor/stemcells in an undifferentiated state.

Example 20

Stem cells are collected from individuals, the cells aretransfected/contacted with the therapeutic vectors, then prepared fortransplantation by standard methods, with or without HLA typing andmatching.

Example 21

Umbilical cord blood samples are obtained from umbilical blood cordbank. The cells are then transfected/contacted with the therapeuticvector of beneficial sequences, then prepared for transplantation bystandard methods, with or without HLA typing and matching. Example 22:Examples of Synthetic oligonucleotide sequences suitable for inclusionin the therapeutic vector.

Any synthetic oligonucleotide sequences that successfully reduce theprotein expression of targeted sequences>70% is covered by the presentinvention.

Any synthetic oligonucleotide sequences that successfully reduce theability of target cells to sustain HIV replication by >70% or to alesser but therapeutic degree or HIV viral activity by >70% or to alesser but therapeutic degree are also covered by this invention.

Examples of miRNA sequences include miRNA sequences derived by IVGNalgorithm(Invitrogen). miRNA sequences targeting the CXCR4 gene includetop strand: 5′-TGCTGATACCAGGCAGGATAAGGCCAGTTTTGGCCACTGACTGACTGGCCTTACTGCCT GGTAT-3′ (SEQ ID NO: 4) and bottom strand:5′-CCTGATACCAGGCAGTAAGGCCAGTCAGTCAGTGGCCAAAACTGGCCTTA TCCTGCCTG GTATC-3′(SEQ ID NO: 5); as well as top strand:5′-TGCTGTGACCAGGATGACCAATCCATGTTTTGGCCACTGACTGACATGGAT TGCATCCTG GTCA-3′(SEQ ID NO: 6) and bottom strand:5′-CCTGTGACCAGGATGCAATCCATGTCAGTCAGTGGCCAAAACATGGATTG GTCATCCTG GTCAC-3′(SEQ ID NO: 7).

Similarly, miRNA sequences targeting the CCR5 gene include top strand:5′-TGCTGATCGGGTGTAAACTGAGCTTGGTTTTGGCCACTGACTGACCAAGCT CATTACACCCGAT-3′(SEQ ID NO: 8) and bottom strand:5′-CCTGATCGGGTGTAATGAGCTTGGTCAGTCAGTGGCCAAAACCAAGCTCA GTTTACACCCGATC-3′;(SEQ ID NO: 9) as well as top strand5′-TGCTGATAGCTTGGTCCAACCTGTTAGTTTTGGCCACTGACTGACTAACAG GTGACCAAGCTAT-3′(SEQ ID NO: 10) and bottom strand:5′-CCTGATAGCTTGGTCACCTGTTAGTCAGTCAGTGGCCAAAACTAACAGGTT GGACCAAGCTATC-3′(SEQ ID NO: 11).

Example 23: Examples of Decoy RNA Suitable for Inclusion in theTherapeutic Vector

Any decoy sequences that successfully reduce the ability of target cellsto sustain ITIV replication by >70% or to a lesser but therapeuticdegree or HIV viral activity by >70% or to a lesser but therapeuticdegree are covered by this invention.

An example TAR decoy sequence is

(SEQ ID NO: 12) gtcgctctgcggagaggctggcagattgagccctgggaggttctctccagcactagcaggtagagcctgggtgttccctgctagactctcaccagtgcttggccggcactgggcagacggctccacgcttgcttgcttaaagacctcttaataaagctgc (Browning et al., 1999)

An example RRE decoy sequence is

(SEQ ID NO: 13) tgctagggttcttgggttttctcgcaacagcaggttctgcaatgggcgcggcgtccctgaccgtgtcggctcagtcccggactttactggccgggatagtgcagcaacagcaacagctgttggacgtggtcaagagacaacaagaactgttgcgactgaccgtctggggaacgaaaaacctccaggcaagagtcactgctatagagaagtacctacaggaccaggcgcggctaaattcatggggatg (Dillon et al., 1990).

Example 24: Flanking Sequences Providing Stability for RNA Decoys

Examples of appropriate flanking sequences for RNA decoys are asfollows:

(SEQ ID NO: 14) GUGCUCGCUUCGGCAGCACGTCGAC---TAR (SEQ ID NO: 15)DECOY SEQ---UCUAGAGCGGACUUCGGUCCGCUUUU (SEQ ID NO: 16)GUGCUCGCUUCGGCAGCACGTCGAC---RRE (SEQ ID NO: 17)DECOY SEQ---UCUAGAGCGGACUUCGGUCCGCUUUU

Previously, it was demonstrated that decoy sequences flanked by hairpinson either side, 19 nucleotides (ntds) of the U6 RNA on the 5′ side aswell as a 3′ stem immediately preceding a poly U terminator for POEIII,showed greater stability. This arrangement is expected to protectagainst 3 ′-5′ exonuclease attack, and to reduce the chances of the 3′trailer interfering with the insert RNA folding. Since only the first3/4 of the tRNA sequence is present, the 5′ end of the insert should beprotected and export from the nucleus should be prevented (Good et al.,1997).

Example 25: Introduction of Therapeutic Vector to the Host

In a preferred embodiment, blood stem/progenitor cells, and target cellsare transfected/contacted with the therapeutic vector(s) (or associatedtherapeutic virus) in vivo by introduction of the therapeutic vector(s)into the host blood, tissues, or bone marrow, etc. The greatest benefitmay be achieved by modifying a large number of endogenous target andstem/progenitor cells. This may be accomplished by using anappropriately-sized, catheter-like device, or needle to inject thetherapeutic vector(s) into the venous or arterial circulation. In apreferred embodiment, the virus is pseudotyped with VSV-G envelopeglycoprotein and native I II V-I env proteins.

Example 26: Introduction of Genetically-Modified Cells into the Host

Blood cells, such as mature peripheral blood T lymphocytes, monocytes,macrophages, T cell progenitors, macrophage-monocyte progenitor cells,and/or pluripotent hematopoietic stem cells (such as those found inumbilical cord blood and occupying bone marrow spaces) as well as otherstem/progenitor cells can be transfected/contacted using the therapeuticvector(s) in vitro. Appropriate concentrations of the therapeuticvector(s) may be those consistent with Browning et al., 1999.Subsequently, cells are expanded (propagated) in vitro, and are thentransferred to the host via introduction of the cells to the venous orarterial circulation using a intravenous needle or catheter.Subsequently, cells transfected/contacted with the therapeutic vectorsare able to “home” to the bone marrow and other tissues.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

Example 27: Examples of Expressed or Targeted Transgenes/ProteinsUtilized in the Present Invention

Any transgene sequences effective in fulfilling the present invention issuitable for use in the present invention. Suitable nucleotide sequencesmay be drawn from any species so long as the desired cells or behavioris achieved. Likewise the method of naming such sequences, either inlower case or upper case letters herein, does not imply a particularspecies. The following sequences stored in the NCBI database (listed byaccession number) represent examples of sequences referenced above inthe present application. They are also examples of specific transgeneencoding sequences (cds) suitable for use in the present invention, butdo not in any way limit the practice of the invention:

(SEQ ID NO: 18)cardiotrophin1.U43030: atgagccggagggagggaagtctggaagacccccagactgattcctcagtctcacttcttccccacttggaggccaagatccgtcagacacacagccttgcgcacctcctcaccaaatacgctgagcagctgctccaggaatatgtgcagctccagggagaccccttcgggctgcccagcttctcgccgccgcggctgccggtggccggcctgagcgccccggctccgagccacgcggggctgccagtgcacgagcggctgcggctggacgcggcggcgctggccgcgctgcccccgctgctggacgcagtgtgtcgccgccaggccgagctgaacccgcgcgcgccgcgcctgctgcgccgcctggaggacgcggcgcgccaggcccgggccctgggcgccgccgtggaggccttgctggccgcgctgggcgccgccaaccgcgggccccgggccgagccccccgccgccaccgcctcagccgcctccgccaccggggtcttccccgccaaggtgctggggctccgcgtttgcggcctctaccgcgagtggctgagccgcaccgagggcgacctgggccagctgctgcccgggggctcggcctga;NM_001330; NM_013246; BC064416; BC036787; BC049822. (SEQ ID NO: 19)CNTF: BC074964: atggctttcacagagcattcaccgctgacccctcaccgtcgggacctctgtagccgctctatctggctagcaaggaagattcgttcagacctgactgctcttacggaatcctatgtgaagcatcagggcctgaacaagaacatcaacctggactctgcggatgggatgccagtggcaagcactgatcagtggagtgagctgaccgaggcagagcgactccaagagaaccttcaagcttatcgtaccttccatgttttgttggccaggctcttagaagaccagcaggtgcattttaccccaaccgaaggtgacttccatcaagctatacatacccttcttctccaagtcgctgcctttgcataccagatagaggagttaatgatactcctggaatacaagatcccccgcaatgaggctgatgggatgcctattaatgttggagatggtggtctctttgagaagaagctgtggggcctaaaggtgctgcaggagctttcacagtggacagtaaggtccatccatgaccttcgtttcatttcttctcatcagactgggatcccagcacgtgggagccattatattgctaacaacaagaaaatgtag; NMJ300614; NM147164; NM_001842. (SEQ ID NO: 20)GP130: NM_175767: atgttgacgttgcagacttggctagtgcaagccttgtttattttcctcaccactgaatctacaggtgaacttctagatccatgtggttatatcagtcctgaatctccagttgtacaacttcattctaatttcactgcagtttgtgtgctaaaggaaaaatgtatggattattttcatgtaaatgctaattacattgtctggaaaacaaaccaltttactattcctaaggagcaatatactatcataaacagaacagcatccagtgtcacctttacagatatagcttcattaaatattcagctcacttgcaacattcttacattcggacagcttgaacagaatgtttatggaatcacaataatttcaggcttgcctccagaaaaacctaaaaatttgagttgcattgtgaacgaggggaagaaaatgaggtgtgagtgggatggtggaagggaaacacacttggagacaaacttcactttaaaatctgaatgggcaacacacaagtttgctgattgcaaagcaaaacgtgacacccccacctcatgcactgttgattattctactgtgtattttgtcaacattgaagtctgggtagaagcagagaatgcccttgggaaggttacatcagatcatatcaattttgatcctgtatataaagtgaagcccaatccgccacataatttatcagtgatcaactcagaggaactgtctagtatcttaaaattgacatggaccaacccaagtattaagagtgttataatactaaaatataacattcaatataggaccaaagatgcctcaacttggagccagattcctcctgaagacacagcatccacccgatcttcattcactgtccaagaccttaaaccttttacagaatatgtgtttaggattcgctgtatgaaggaagatggtaagggatactggagtgactggagtgaagaagcaagtgggatcacctatgaagataacattgcctcctttlga; NM_002184;EF442778. (SEQ ID NO: 21) IL6: BC015511:atgaactccttctccacaagcgccttcggtccagttgccttctccctggggctgctcctggtgttgcctgctgccttccctgccccagtacccccaggagaagattccaaagatgtagccgccccacacagacagccactcacctcttcagaacgaattgacaaacaaattcggtacatcctcgacggcatctcagccctgagaaaggagacatgtaacaagagtaacatgtgtgaaagcagcaaagaggcactggcagaaaacaacctgaaccttccaaagatggctgaaaaagatggatgcttccaatctggattcaatgaggagacttgcctggtgaaaatcatcactggtcttttggagtttgaggtatacctagagtacctccagaacagatttgagagtagtgaggaacaagccagagctgtgcagatgagtacaaaagtcctgatccagttcctgcagaaaaaggcaaagaatctagatgcaataaccacccctgacccaaccacaaatgccagcctgctgacgaagctgcaggcacagaaccagtggctgcaggacatgacaactcatctcattctgcgcagctttaaggagttcctgcagtccagcctgagggctcttcggcaaatgtag; AB 107656.(SEQ ID NO: 22)HOXB4: NM_024015: atggctatgagttcttttttgatcaactcaaactatgtcgaccccaagttccctccatgcgaggaatattcacagagcgaltacctacccagcgaccactcgcccgggtactacgccggcggccagaggcgagagagcagcttccagccggaggcgggctlcgggcggcgcgcggcgtgcaccgtgcagcgctacgcggcctgccgggaccctgggcccccgccgcctccgccaccacccccgccgcccccgccaccgcccggtctgtcccctcgggctcctgcgccgccacccgccggggccctcctcccggagcccggccagcgctgcgaggcggtcagcagcagccccccgccgcctccctgcgcccagaaccccctgcaccccagcccgtcccactccgcgtgcaaagagcccgtcgtctacccctggatgcgcaaagttcacgtgagcacggtaaaccccaattacgccggcggggagcccaagcgctctcggaccgcctacacgcgccagcaggtcttggagctggagaaggaatttcactacaaccgctacctgacacggcgccggagggtggagatcgcccacgcgctctgcctctccgagcgccagatcaagatctggttccagaaccggcgcatgaagtggaaaaaagaccacaagttgcccaacaccaagatccgctcgggtggtgcggcaggctcagccggagggccccctggccggcccaatggaggcccccgcgcgctctag; NM_010459.(SEQ ID NO: 23)IL6R: NM_000565: atgctggccgtcggctgcgcgctgctggctgccctgctggccgcgccgggagcggcgctggccccaaggcgctgccctgcgcaggaggtggcgagaggcgtgctgaccagtctgccaggagacagcgtgactctgacctgcccgggggtagagccggaagacaatgccactgttcactgggtgctcaggaagccggctgcaggctcccaccccagcagatgggctggcatgggaaggaggctgctgctgaggtcggtgcagctccacgactctggaaactattcatgctaccgggccggccgcccagctgggactgtgcacttgctggtggatgttccccccgaggagccccagctctcctgcttccggaagagccccctcagcaatgttgtttgtgagtggggtcctcggagcaccccatccctgacgacaaaggctgtgctcttggtgaggaagtltcagaacagtccggccgaagacttccaggagccgtgccagtattcccaggagtcccagaagtlctcctgccagttagcagtcccggagggagacagctctttctacatagtgtccatgtgcgtcgccagtagtgtcgggagcaagttcagcaaaactcaaacctttcagggttgtggaatcttgcagcctgatccgcctgccaacatcacagtcactgccgtggccagaaacccccgctggctcagtgtcacctggcaagacccccactcctggaactcatctttctacagactacggtttgagctcagatatcgggctgaacggtcaaagacattcacaacatggatggtcaaggacctccagcatcactgtgtcatccacgacgcctggagcggcctgaggcacgtgglgcagcttcgtgcccaggaggagttcgggcaaggcgagtggagcgagtggagcccggaggccatgggcacgccttggacagaatccaggagtcctccagctgagaacgaggtgtccacccccatgcaggcacttactactaataaagacgatgataatattctcttcagagattctgcaaatgcgacaagcctcccagtgcaagattcttcttcagtaccactgcccacattcctggttgctggagggagcctggccttcggaacgctcctctgcattgccattgttctgaggttcaagaagacgtggaagctgcgggctctgaaggaaggcaagacaagcatgcatccgccgtactctttggggcagctggtcccggagaggcctcgacccaccccagtgcttgttcctctcatctccccaccggtgtcccccagcagcctggggtctgacaatacctcgagccacaaccgaccagatgccagggacccacggagcccttatgacatcagcaatacagactacttcttccccagatag; NM_181359.(SEQ ID NO: 24) IL11: NM_133519:atgaactgtgtttgtcgcctggtcctggtggtgctgagcctctggccagatagagtcgttgcccctgggccaccagctggctcccctcgagtgtcttcagaccctcgtgcagatctggatagcgctgtcctcttgaccaggtccctcctggcagacacacggcaactagctgcacagatgagagacaaattcccagctgatggagaccacaatctggactccctacctaccttggccatgagcgctgggacactgggatctttgcagcttcctggagtgctgacaaggcttcgagtagacttaatgtcctacttccgacatgtacagtggttgcgccgggcagctggtccttccctaaagactctggagccagagctgggtgccctgcaagcccgactggaacggctacttcgtcgcttacagctcttgatgtctcgcctagccttgccccaggcagccccggaccaacctgcggtccctctgggccctcctgcctcggcctggggaagcatccgggcagctcatgccatcctaggagggctgcacctgaccttggactgggccgtgcggggcctgctgttgttaaagactcggctgtaa; NM_008350. (SEQ ID NO: 25)LIF: NM_002309: atgaaggtcttggcggcaggagttgtgcccctgctgttggttctgcactggaaacatggggcggggagccccctccccatcacccctgtcaacgccacctgtgccatacgccacccatgtcacaacaacctcatgaaccagatcaggagccaactggcacagctcaatggcagtgccaatgccctctttattctctattacacagcccagggggagccgttccccaacaacctggacaagctatgtggccccaacgtgacggacttcccgcccttccacgccaacggcacggagaaggccaagctggtggagctgtaccgcatagtcgtgtaccttggcacctccctgggcaacatcacccgggaccagaagatcctcaaccccagtgccctcagcctccacagcaagctcaacgccaccgccgacatcctgcgaggcctccttagcaacgtgctgtgccgcctgtgcagcaagtaccacgtgggccatgtggacgtgacctacggccctgacacctcgggtaaggatgtcttccagaagaagaagctgggctgtcaactcctggggaagtataagcagatcatcgccgtgttggcccaggccttctag; NM_008501; BB235045.(SEQ ID NO: 26)LIFR: NM_ 002310: atgatggatatttacgtatgtttgaaacgaccatcctggatggtggacaataaaagaatgaggactgcttcaaatttccagtggctgttatcaacatttattcttctatatctaatgaatcaagtaaatagccagaaaaagggggctcctcatgatttgaagtgtgtaactaacaatttgcaagtgtggaactgttcttggaaagcaccctctggaacaggccgtggtactgattatgaagtttgcattgaaaacaggtcccgttcttgttatcagttggagaaaaccagtattaaaattccagctctttcacatggtgattatgaaataacaataaattctctacatgattttggaagttctacaagtaaattcacactaaatgaacaaaacgtttccttaattccagatactccagagatcttgaatttgtctgctgatttctcaacctctacattatacctaaagtggaacgacaggggttcagtttttccacaccgctcaaatgttatctgggaaattaaagttctacgtaaagagagtatggagctcgtaaaattagtgacccacaacacaactctgaatggcaaagatacacttcatcactggagttgggcctcagatatgcccttggaatgtgccattcattttgtggaaattagatgctacattgacaatcttcatttttctggtctcgaagagtggagtgactggagccctgtgaagaacatttcttggatacctgattctcagactaaggtttttcctcaagataaagtgatacttgtaggctcagacataacattttgttgtgtgagtcaagaaaaagtgttatcagcactgattggccatacaaactgccccttgatccatcttgatggggaaaatgttgcaatcaagattcgtaatatttctgtttctgcaagtagtggaacaaatgtagtttttacaaccgaagataacatatttggaaccgttatttttgctggatatccaccagatactcctcaacaactgaattgtgagacacatgatttaaaagaaattatatgtagttggaatccaggaagggtgacagcgttggtgggcccacgtgctacaagctacactttagttgaaagtttttcaggaaaatatgttagacttaaaagagctgaagcacctacaaacgaaagctatcaattattatttcaaatgcttccaaatcaagaaatatataattttactttgaatgctcacaatccgctgggtcgatcacaatcaacaattttagttaatataactgaaaaagtttatccccatactcctacttcattcaaagtgaaggatattaattcaacagctgttaaactttcttggcatttaccaggcaactttgcaaagattaatlttttatgtgaaattgaaattaagaaatctaattcagtacaagagcagcggaatgtcacaatcaaaggagtagaaaattcaagttatcttgttgctctggacaagttaaatccatacactctatatacttttcggattcgttgttctactgaaactttctggaaatggagcaaatggagcaataaaaaacaacatttaacaacagaagccagtccttcaaaggggcctgatacttggagagagtggagttctgatggaaaaaatttaataatctattggaagcctttacccattaatgaagctaatggaaaaatactttcctacaatgtatcgtgttcatcagatgaggaaacacagtccctttctgaaatccctgatcctcagcacaaagcagagatacgacttgataagaatgactacatcatcagcgtagtggctaaaaattctgtgggctcatcaccaccttccaaaatagcgagtatggaaattccaaatgatgatctcaaaatagaacaagttgttgggatgggaaaggggattctcctcacctggcattacgaccccaacatgacttgcgactacgtcattaagtggtgtaactcgtctcggtcggaaccatgccttatggactggagaaaagttccctcaaacagcactgaaactgtaatagaatctgatgagtttcgaccaggtataagatataattttttcctgtatggatgcagaaatcaaggatatcaattattacgctccatgattggatatatagaagaattggctcccattgttgcaccaaattttactgttgaggatacttctgcagattcgatattagtaaaatgggaagacattcctgtggaagaacttagaggctttttaagaggatatttgttttactttggaaaaggagaaagagacacatctaagatgagggttttagaatcaggtcgttctgacataaaagttaagaatattactgacatatcccagaagacactgagaattgctgatcttcaaggtaaaacaagttaccacctggtcttgcgagcctatacagatggtggagtgggcccggagaagagtatgtatgtggtgacaaaggaaaattctgtgggattaattattgccattctcatcccagtggcagtggctgtcattgttggagtggtgacaagtatccttlgctatcggaaacgagaatggattaaagaaaccttctaccctgatattccaaatccagaaaactgtaaagcattacagtttcaaaagagtgtctgtgagggaagcagtgctcttaaaacattggaaatgaatccttgtaccccaaataatgttgaggttctggaaactcgatcagcatttcctaaaatagaagatacagaaataatttccccagtagctgagcgtcctgaagatcgctctgatgcagagcctgaaaaccatgtggttgtgtcctattgtccacccatcattgaggaagaaataccaaacccagccgcagatgaagctggagggactgcacaggttatttacattgatgttcagtcgatgtatcagcctcaagcaaaaccagaagaagaacaagaaaatgaccctgtaggaggggcaggctataagccacagatgcacctccccattaattctactgtggaagatatagctgcagaagaggacttagataaaactgcgggttacagacctcaggccaatgtaaatacatggaatttagtgtctccagactctcctagatccatagacagcaacagtgagattgtctcatttggaagtccatgctccattaatlcccgacaatttttgattcctcctaaagatgaagactctcctaaatctaatggaggagggtggtcctttacaaacttttttcagaacaaaccaaacgattaa; NM 013584; NM_031048. (SEQ ID NO: 27) STAT3: NM_003 150:atggcccaatggaatcagctacagcagcttgacacacggtacctggagcagctccatcagctctacagtgacagcttcccaatggagctgcggcagtttctggccccttggattgagagtcaagattgggcatatgcggccagcaaagaatcacatgccactttggtgtttcataatctcctgggagagattgaccagcagtatagccgcttcctgcaagagtcgaatgttctctatcagcacaatctacgaagaatcaagcagtttcttcagagcaggtatcttgagaagccaatggagattgcccggattgtggcccggtgcctgtgggaagaatcacgccttctacagactgcagccactgcggcccagcaagggggccaggccaaccaccccacagcagccgtggtgacggagaagcagcagatgctggagcagcaccttcaggatgtccggaagagagtgcaggatctagaacagaaaatgaaagtggtagagaatctccaggatgactttgatttcaactataaaaccctcaagagtcaaggagacatgcaagatctgaatggaaacaaccagtcagtgaccaggcagaagatgcagcagctggaacagatgctcactgcgctggaccagatgcggagaagcatcgtgagtgagctggcggggcttttgtcagcgatggagtacgtgcagaaaactctcacggacgaggagctggctgactggaagaggcggcaacagattgcctgcattggaggcccgcccaacatctgcctagatcggctagaaaactggataacgtcattagcagaatctcaacttcagacccgtcaacaaattaagaaactggaggagttgcagcaaaaagtttcctacaaaggggaccccattgtacagcaccggccgatgctggaggagagaatcgtggagctgtttagaaacttaatgaaaagtgcctttgtggtggagcggcagccctgcatgcccatgcatcctgaccggcccctcgtcatcaagaccggcgtccagttcactactaaagtcaggttgctggtcaaattccctgagttgaattatcagcttaaaattaaagtgtgcattgacaaagactctggggacgttgcagctctcagaggatcccggaaatttaacattctgggcacaaacacaaaagtgatgaacatggaagaatccaacaacggcagcctctctgcagaattcaaacacttgaccctgagggagcagagatgtgggaatgggggccgagccaattgtgatgcttccctgattgtgactgaggagctgcacctgatcacctttgagaccgaggtgtatcaccaaggcctcaagattgacctagagacccactccttgccagttgtggtgatctccaacatctgtcagatgccaaatgcctgggcgtccatcctgtggtacaacatgctgaccaacaatcccaagaatgtaaacttttttaccaagcccccaattggaacctgggatcaagtggccgaggtcctgagctggcagttctcctccaccaccaagcgaggactgagcatcgagcagctgactacactggcagagaaactcttgggacctggtgtgaattattcagggtgtcagatcacatgggctaaattttgcaaagaaaacatggctggcaagggcttctccttctgggtctggctggacaatatcattgaccttgtgaaaaagtacatcctggccctttggaacgaagggtacatcatgggctttatcagtaaggagcgggagcgggccatcttgagcactaagcctccaggcacctlcctgctaagattcagtgaaagcagcaaagaaggaggcgtcactttcacttgggtggagaaggacatcagcggtaagacccagatccagtccgtggaaccatacacaaagcagcagctgaacaacatgtcatttgctgaaatcatcatgggctataagatcatggatgctaccaatatcctggtgtctccactggtctatctctatcctgacattcccaaggaggaggcattcggaaagtattgtcggccagagagccaggagcatcctgaagctgacccaggcgctgccccatacctgaagaccaagtttatctgtgtgacaccaacgacctgcagcaataccattgacctgccgatgtccccccgcactttagattcattgatgcagtttggaaataatggtgaaggtgctgaaccctcagcaggagggcagtttgagtccctcacctttgacatggagttgacctcggagtgcgctacctcccccatgtga; NMJ 13662; NM_139276. (SEQ ID NO: 28)NUMB: AF171938:atgaacaaattacggcaaagttttaggagaaagaaggatgtttatgttccagaggccagtcgtccacatcagtggcagacagatgaagaaggcgttcgcaccggaaaatgtagcttcccggttaagtaccttggccatgtagaagttgatgaatcaagaggaatgcacatctgtgaagatgctgtaaaaagattgaaagctgaaaggaagttcttcaaaggcttctttggaaaaactggaaagaaagcagttaaagcagttctgtgggtctcagcagatggactcagagttgtggatgaaaaaactaaggacctcatagttgaccagacgatagagaaagtttctttctgtgccccagacaggaactttgatagagccttttcttacatatgccgtgatggcaccactcgtcgctggatctgtcactgcttcatggctgtcaaggacacaggtgaaaggttgagccatgcagtaggctgtgcttttgcagcctgtttagagcgcaagcagaagcgggagaaggaatgtggagtgactgctacttttgatgctagtcggaccacttttacaagagaaggatcattccgtgtcacaacagccactgaacaagcagaaagagaggagatcatgaaacaaatgcaagatgccaagaaagctgaaacagataagatagtcgttggttcatcagttgcccctggcaacactgccccatccccatcctctcccacctctcctacttctgatgccacgacctctctggagatgaacaatcctcatgccatcccacgccggcatgctccaattgaacagcttgctcgccaaggctctttccgaggttttcctgctcttagccagaagatgtcaccctttaaacgccaactatccctacgcatcaatgagttgccttccactatgcagaggaagactgatttccccattaaaaatgcagtgccagaagtagaaggggaggcagagagcatcagctccctgtgctcacagatcaccaatgccttcagcacacctgaggaccccttctcatctgctccgatgaccaaaccagtgacagtggtggcaccacaatctcctaccttccaagctaatggcactgactcagccttccatgtgcttgctaagccagcccatactgctctagcacccgtagcaatgcctgtgcgtgaaaccaacccttgggcccatgcccctgatgctgctaacaaggaaattgcagccacatgttcggggaccgagtggggtcaatcttctggtgctgcctctccaggtctcttccaggccggtcatagacgtactccctctgaggccgaccgatggttagaagaggtgtctaagagcgtccgggctcagcagccccaggcctcagctgctcctctgcagccagttctccagcctcctccacccactgccatctcccagccagcatcacctttccaagggaatgcattcctcacctctcagcctgtgccagtgggtgtggtcccagccctgcaaccagcctttgtccctgcccagtcctatcctgtggccaatggaatgccctatccagcccctaatgtgcctgtggtgggcatcactccctcccagatggtggccaacgtatttggcactgcaggccaccctcaggctgcccatccccatcagtcacccagcctggtcaggcagcagacattccctcactacgaggcaagcagtgctaccaccagtcccttctttaagcctcctgctcagcacctcaacggttctgcagctttcaatggtgtagatgatggcaggttggcctcagcagacaggcatacagaggttcctacaggcacctgcccagtggatccttttgaagcccagtgggctgcattagaaaataagtccaagcagcgtactaatccctcccctaccaaccctttctccagtgacttacagaagacgtttgaaattgaactttaa;(SEQ ID NO: 29)AF171939: atgaacaaattacggcaaagttttaggagaaagaaggatgtttatgttccagaggccagtcgtccacatcagtggcagacagatgaagaaggcgttcgcaccggaaaatgtagcttcccggttaagtaccttggccatgtagaagttgatgaatcaagaggaatgcacatctgtgaagatgctgtaaaaagattgaaagctgaaaggaagttcttcaaaggcttctttggaaaaactggaaagaaagcagttaaagcagttctgtgggtctcagcagatggactcagagttgtggatgaaaaaactaaggacctcatagttgaccagacgatagagaaagtttctttctgtgccccagacaggaactttgatagagccttttcttacatatgccgtgatggcaccactcgtcgctggatctgtcactgcttcatggctgtcaaggacacaggtgaaaggttgagccatgcagtaggctgtgcttttgcagcctgtttagagcgcaagcagaagcgggagaaggaatgtggagtgactgctacttttgatgctagtcggaccacttttacaagagaaggatcattccgtgtcacaacagccactgaacaagcagaaagagaggagatcatgaaacaaatgcaagatgccaagaaagctgaaacagataagatagtcgttggttcatcagttgcccctggcaacactgccccatccccatcctctcccacctctcctacttctgatgccacgacctctctggagatgaacaatcctcatgccatcccacgccggcatgctccaattgaacagcttgctcgccaaggctctttccgaggttttcctgctcttagccagaagatgtcaccctttaaacgccaactatccctacgcatcaatgagttgccttccactatgcagaggaagactgatttccccattaaaaatgcagtgccagaagtagaaggggaggcagagagcatcagctccctgtgctcacagatcaccaatgccttcagcacacctgaggaccccttctcatctgctccgatgaccaaaccagtgacagtggtggcaccacaatctcctaccttccaagggaccgagtggggtcaatcttctggtgctgcctctccaggtctcttccaggccggtcatagacgtactccctctgaggccgaccgatggttagaagaggtgtctaagagcgtccgggctcagcagccccaggcctcagctgctcctctgcagccagttctccagcctcctccacccactgccatctcccagccagcatcacctttccaagggaatgcattcctcacctctcagcctgtgccagtgggtgtggtcccagccctgcaaccagcctttgtccctgcccagtcctatcctgtggccaatggaatgccctatccagcccctaatgtgcctgtggtgggcatcactccctcccagatggtggccaacgtatttggcactgcaggccaccctcaggctgcccatccccatcagtcacccagcctggtcaggcagcagacattccctcactacgaggcaagcagtgctaccaccagtcccttctttaagcctcctgctcagcacctcaacggttctgcagctttcaatggtgtagatgatggcaggttggcctcagcagacaggcatacagaggttcctacaggcacctgcccagtggatccttttgaagcccagtgggctgcattagaaaataagtccaagcagcgtactaatccctcccctaccaaccctttctccagtgacttacagaagacgtttgaaattgaactttaa; (SEQ ID NO: 30) AF171940:atgaacaaattacggcaaagttttaggagaaagaaggatgtttatgttccagaggccagtcgtccacatcagtggcagacagatgaagaaggcgttcgcaccggaaaatgtagcttcccggttaagtaccttggccatgtagaagttgatgaatcaagaggaatgcacatctgtgaagatgctgtaaaaagattgaaagctactggaaagaaagcagttaaagcagttctgtgggtctcagcagatggactcagagttgtggatgaaaaaactaaggacctcatagttgaccagacgatagagaaagtttctttctgtgccccagacaggaactttgatagagccttttcttacatatgccgtgatggcaccactcgtcgctggatctgtcactgcttcatggctgtcaaggacacaggtgaaaggttgagccatgcagtaggctgtgcttttgcagcctgtttagagcgcaagcagaagcgggagaaggaatgtggagtgactgctacttttgatgctagtcggaccacttttacaagagaaggatcattccgtgtcacaacagccactgaacaagcagaaagagaggagatcatgaaacaaatgcaagatgccaagaaagctgaaacagataagatagtcgttggttcatcagttgcccctggcaacactgccccatccccatcctctcccacctctcctacttctgatgccacgacctctctggagatgaacaatcctcatgccatcccacgccggcatgctccaattgaacagcttgctcgccaaggctctttccgaggttttcctgctcttagccagaagatgtcaccctttaaacgccaactatccctacgcatcaatgagttgccttccactatgcagaggaagactgatttccccattaaaaatgcagtgccagaagtagaaggggaggcagagagcatcagctccctgtgctcacagatcaccaatgccttcagcacacctgaggaccccttctcatctgctccgatgaccaaaccagtgacagtggtggcaccacaatctcctaccttccaagctaatggcactgactcagccttccatgtgcttgctaagccagcccatactgctctagcacccgtagcaatgcctgtgcgtgaaaccaacccttgggcccatgcccctgatgctgctaacaaggaaattgcagccacatgttcggggaccgagtggggtcaatcttctggtgctgcctctccaggtctcttccaggccggtcatagacgtactccctctgaggccgaccgatggttagaagaggtgtctaagagcgtccgggctcagcagccccaggcctcagctgctcctctgcagccagttctccagcctcctccacccactgccatctcccagccagcatcacctttccaagggaatgcattcctcacctctcagcctgtgccagtgggtgtggtcccagccctgcaaccagcctttgtccctgcccagtcctatcctgtggccaatggaatgccctatccagcccctaatgtgcctgtggtgggcatcactccctcccagatggtggccaacgtatttggcactgcaggccaccctcaggctgcccatccccatcagtcacccagcctggtcaggcagcagacattccctcactacgaggcaagcagtgctaccaccagtcccttctltaagcctcctgctcagcacctcaacggttctgcagctttcaatggtgtagatgatggcaggttggcctcagcagacaggcatacagaggtlcctacaggcacctgcccagtggatccttttgaagcccagtgggctgcattagaaaataagtccaagcagcgtactaatccctcccctaccaaccctttctccagtgacttacagaagacgtttgaaattgaactttaa; (SEQ ID NO: 31) AF171941:atgaacaaattacggcaaagttttaggagaaagaaggatgtttatgttccagaggccagtcgtccacatcagtggcagacagatgaagaaggcgttcgcaccggaaaatgtagcttcccggttaagtaccttggccatgtagaagttgatgaatcaagaggaatgcacatctgtgaagatgctgtaaaaagattgaaagctactggaaagaaagcagttaaagcagttctgtgggtctcagcagatggactcagagttgtggatgaaaaaactaaggacctcatagttgaccagacgatagagaaagtttctttctgtgccccagacaggaactttgatagagccttttcttacatatgccgtgatggcaccactcgtcgctggatctgtcactgcttcatggctgtcaaggacacaggtgaaaggttgagccatgcagtaggctgtgcttttgcagcctgtttagagcgcaagcagaagcgggagaaggaatgtggagtgactgctacttttgatgctagtcggaccacttttacaagagaaggatcattccgtgtcacaacagccactgaacaagcagaaagagaggagatcatgaaacaaatgcaagatgccaagaaagctgaaacagataagatagtcgttggttcatcagttgcccctggcaacactgccccatccccatcctctcccacctctcctacttctgatgccacgacctctctggagatgaacaatcctcatgccatcccacgccggcatgctccaattgaacagcttgctcgccaaggctctttccgaggttttcctgctcttagccagaagatgtcaccctttaaacgccaactatccctacgcatcaatgagttgccttccactatgcagaggaagactgatttccccattaaaaatgcagtgccagaagtagaaggggaggcagagagcatcagctccctgtgctcacagatcaccaatgccttcagcacacctgaggaccccttctcatctgctccgatgaccaaaccagtgacagtggtggcaccacaatctcctaccttccaagggaccgagtggggtcaatcttctggtgctgcctctccaggtctcttccaggccggtcatagacgtactccctctgaggccgaccgatggttagaagaggtgtctaagagcgtccgggctcagcagccccaggcctcagctgctcctctgcagccagttctccagcctcctccacccactgccatctcccagccagcatcacctttccaagggaatgcattcctcacctctcagcctgtgccagtgggtgtggtcccagccctgcaaccagcctttgtccctgcccagtcctatcctgtggccaatggaatgccctatccagcccctaatgtgcctgtggtgggcatcactccctcccagatggtggccaacgtatttggcactgcaggccaccctcaggctgcccatccccatcagtcacccagcctggtcaggcagcagacattccctcactacgaggcaagcagtgctaccaccagtcccttctttaagcctcctgctcagcacctcaacggttctgcagctttcaatggtgtagatgatggcaggttggcctcagcagacaggcatacagaggttcctacaggcacctgcccagtggatccttttgaagcccagtgggctgcattagaaaataagtccaagcagcgtactaatccctcccctaccaaccctttctccagtgacttacagaagacgtttgaaattgaactttaa; NMJ 10949; NMJ 33287;BB483123; NM 010950; NM_010949; NM 004756; DQ022744. (SEQ ID NO: 32)Numblike: NM 00475:atgtcccgcagcgcggcggccagcggcggaccccggaggcctgagcggcacctgcccccagccccctgtggggccccggggcccccagaaacctgcaggacggagccagacggggcgggcaccatgaacaagttacggcagagcctgcggcggaggaagccagcctacgtgcccgaggcgtcgcgcccgcaccagtggcaggcagacgaggacgcggtgcggaagggcacgtgcagcttcccggtcaggtacctgggtcacgtggaggtagaggagtcccggggaatgcacgtgtgtgaagatgcggtgaagaagctgaaggcgatgggccgaaagtccgtgaagtctgtcctgtgggtgtcagccgatgggctccgagtggtggacgacaaaaccaaggatcttctggtcgaccagaccatcgaaaaggtctccttttgtgctcctgaccgcaacctggacaaggctttctcctatatctgtcgtgacgggactacccgccgctggatctgccactgttttctggcactgaaggactccggcgagaggctgagccacgctgtgggctgtgcttttgccgcctgcctggagcgaaaacagcgacgggagaaggaatgtggggtcacggccgccttcgatgccagccgcaccagcttcgcccgcgagggctccttccgcctgtctgggggtgggcggcctgctgagcgagaggccccggacaagaagaaagcagaggcagcagctgcccccactgtggctcctggccctgcccagcctgggcacgtgtccccgacaccagccaccacatcccctggtgagaagggtgaggcaggcacccctgtggctgcaggcaccactgcggccgccatcccccggcgccatgcacccctggagcagctggttcgccagggctccttccgtgggttcccagcactcagccagaagaactcgcctttcaaacggcagctgagcctacggctgaatgagctgccatccacgctgcagcgccgcactgacttccaggtgaagggcacagtgcctgagatggagcctcctggtgccggcgacagtgacagcatcaacgctctgtgcacacagatcagttcatcttttgccagtgctggagcgccagcaccagggccaccacctgccacaacagggacttctgcctggggtgagccctccgtgccccctgcagctgccttccagcctgggcacaagcggacaccttcagaggctgagcgatggctggaggaggtgtcacaggtggccaaggcccagcagcagcagcagcagcaacagcaacagcagcagcagcagcagcagcaacagcagcaagcagcctcagtggccccagtgcccaccatgcctcctgccctgcagcctltccccgcccccgtggggccctttgacgctgcacctgcccaagtggccgtgttcctgccacccccacacatgcagcccccttttgtgcccgcctacccgggcttgggctacccaccgatgccccgggtgcccgtggtgggcatcacaccctcacagatggtggcaaacgccttctgctcagccgcccagctccagcctcagcctgccactctgcttgggaaagctggggccttcccgccccctgccatacccagtgcccctgggagccaggcccgccctcgccccaatggggccccctggccccctgagccagcgcctgccccagctccagagttggacccctttgaggcccagtgggcggcattagaaggcaaagccactgtagagaaaccctccaaccccttttctggcgacctgcaaaagacattcgagattgaactgtag; U96441; NM O 10950; DQ022744. (SEQ ID NO: 33) NANOG: NM 024865:atgagtgtggatccagcttgtccccaaagcttgccttgctttgaagcatccgactgtaaagaatcttcacctatgcctgtgatttgtgggcctgaagaaaactatccatccttgcaaatgtcttctgctgagatgcctcacacggagactgtctctcctcttccttcctccatggatctgcttattcaggacagccctgattcttccaccagtcccaaaggcaaacaacccacttctgcagagaagagtgtcgcaaaaaaggaagacaaggtcccggtcaagaaacagaagaccagaactgtgttctcttccacccagctgtgtgtactcaatgatagatttcagagacagaaatacctcagcctccagcagatgcaagaactctccaacatcctgaacctcagctacaaacaggtgaagacctggttccagaaccagagaatgaaatctaagaggtggcagaaaaacaactggccgaagaatagcaatggtgtgacgcagaaggcctcagcacctacctaccccagcctttactcttcctaccaccagggatgcctggtgaacccgactgggaaccttccaatgtggagcaaccagacctggaacaattcaacctggagcaaccagacccagaacatccagtcctggagcaaccactcctggaacactcagacctggtgcacccaatcctggaacaatcaggcctggaacagtcccttctataactgtggagaggaatctctgcagtcctgcatgcagttccagccaaattctcctgccagtgacttggaggctgccttggaagctgctggggaaggccttaatgtaatacagcagaccactaggtattttagtactccacaaaccatggatttattcctaaactactccatgaacatgcaacctgaagacgtgtga; BC 137873; NM 028016; NM_013633; BC160187. (SEQ ID NO: 34)OncostatinM(OSM): NM_020530: atgggggtactgctcacacagaggacgctgctcagtctggtccttgcactcctgtttccaagcatggcgagcatggcggctataggcagctgctcgaaagagtaccgcgtgctccttggccagctccagaagcagacagatctcatgcaggacaccagcagactcctggacccctatatacgtatccaaggcctggatgttcctaaactgagagagcactgcagggagcgccccggggccttccccagtgaggagaccctgagggggctgggcaggcggggcttcctgcagaccctcaatgccacactgggctgcgtcctgcacagactggccgacttagagcagcgcctccccaaggcccaggatttggagaggtctgggctgaacatcgaggacttggagaagctgcagatggcgaggccgaacatcctcgggctcaggaacaacatctactgcatggcccagctgctggacaactcagacacggctgagcccacgaaggctggccggggggcctctcagccgcccacccccacccctgcctcggatgcttttcagcgcaagctggagggctgcaggttcctgcatggctaccatcgcttcatgcactcagtggggcgggtcttcagcaagtggggggagagcccgaaccggagccggagacacagcccccaccaggccctgaggaagggggtgcgcaggaccagaccctccaggaaaggcaagagactcatgaccaggggacagctgccccggtag;NM_001013365; NP_065391; NP_001013383. (SEQ ID NO: 35)OSMR: NM_003999: atggctctatttgcagtctttcagacaacattcttcttaacattgctgtccttgaggacttaccagagtgaagtcttggctgaacgtttaccattgactcctgtatcacttaaagtttccaccaattctacgcgtcagagtttgcacttacaatggactgtccacaaccttccttatcatcaggaattgaaaatggtatttcagatccagatcagtaggattgaaacatccaatgtcatctgggtggggaattacagcaccactgtgaagtggaaccaggttctgcattggagctgggaatctgagctccctttggaatgtgccacacactttgtaagaataaagagtttggtggacgatgccaagttccctgagccaaatttctggagcaactggagttcctgggaggaagtcagtgtacaagattctactggacaggatatattgttcgttttccctaaagataagctggtggaagaaggcaccaatgttaccatttgttacgtttctaggaacattcaaaataatgtatcctgttatttggaagggaaacagattcatggagaacaacttgatccacatgtaactgcattcaacttgaatagtgtgcctttcattaggaataaagggacaaatatctattgtgaggcaagtcaaggaaatgtcagtgaaggcatgaaaggcatcgttctttttgtctcaaaagtacttgaggagcccaaggacttttcttgtgaaaccgaggacttcaagactttgcactgtacttgggatcctgggacggacactgccttggggtggtctaaacaaccttcccaaagctacactttatttgaatcattttctggggaaaagaaactttgtacacacaaaaactggtgtaattggcaaataactcaagactcacaagaaacctataacttcacactcatagctgaaaattacttaaggaagagaagtgtcaatatcctttttaacctgactcatcgagtttatttaatgaatccttttagtgtcaactttgaaaatgtaaatgccacaaatgccatcatgacctggaaggtgcactccataaggaataatttcacatatttgtgtcagattgaactccatggtgaaggaaaaatgatgcaatacaatgtttccatcaaggtgaacggtgagtacttcttaagtgaactggaacctgccacagagtacatggcgcgagtacggtgtgctgatgccagccacttctggaaatggagtgaatggagtggtcagaacttcaccacacttgaagctgctccctcagaggcccctgatgtctggagaattgtgagcttggagccaggaaatcatactgtgaccttattctggaagccattatcaaaactgcatgccaatggaaagatcctgttctataatgtagttgtagaaaacctagacaaaccatccagttcagagctccattccattccagcaccagccaacagcacaaaactaatccttgacaggtgttcctaccaaatctgcgtcatagccaacaacagtgtgggtgcttctcctgcttctgtaatagtcatctctgcagaccccgaaaacaaagaggttgaggaagaaagaattgcaggcacagagggtggattctctctgtcttggaaaccccaacctggagatgttataggctatgttgtggactgglgtgaccatacccaggatgtgctcggtgatttccagtggaagaatgtaggtcccaataccacaagcacagtcattagcacagatgcttttaggccaggagttcgatatgacttcagaatttatgggttatctacaaaaaggattgcttgtttattagagaaaaaaacaggatactctcaggaacttgctccttcagacaaccctcacgtgctggtggatacattgacatcccactccttcactctgagttggaaagattactctactgaatctcaacctggttttatacaagggtaccatgtctatctgaaatccaaggcgaggcagtgccacccacgatttgaaaaggcagttctttcagatggttcagaatgttgcaaatacaaaattgacaacccggaagaaaaggcattgattgtggacaacctaaagccagaatccttctatgagtttttcatcactccattcactagtgctggtgaaggccccagtgctacgttcacgaaggtcacgactccggatgaacactcctcgatgctgattcatatcctactgcccatggttttctgcgtcttgctcatcatggtcatgtgctacttgaaaagtcagtggatcaaggagacctgttatcctgacatccctgacccttacaagagcagcatcctgtcattaataaaattcaaggagaaccctcacctaataataatgaatgtcagtgactgtatcccagatgctattgaagttgtaagcaagccagaagggacaaagatacagttcctaggcactaggaagtcactcacagaaaccgagttgactaagcctaactacctttatctccttccaacagaaaagaatcactctggccctggcccctgcatctgttttgagaacttgacctataaccaggcagcttctgactctggctcttgtggccatgttccagtatccccaaaagccccaagtatgctgggactaatgacctcacctgaaaatgtactaaaggcactagaaaaaaactacatgaactccctgggagaaatcccagctggagaaacaagtttgaattatgtgtcccagttggcttcacccatgtttggagacaaggacagtctcccaacaaacccagtagaggcaccacactgttcagagtataaaatgcaaatggcagtctccctgcgtcttgccttgcctcccccgaccgagaatagcagcctctcctcaattacccttttagatccaggtgaacactactgctaa; NP_003990.1. (SEQ ID NO: 36)OCT3/4(POU5F1): NIVI_203289: atgcacttctacagactattccttggggccacacgtaggttcttgaatcccgaatggaaaggggagattgataactggtgtgtttatgttcttacaagtcttctgccttttaaaatccagtcccaggacatcaaagctctgcagaaagaactcgagcaatttgccaagctcctgaagcagaagaggatcaccctgggatatacacaggccgatgtggggctcaccctgggggttctatttgggaaggtattcagccaaacgaccatctgccgctltgaggctctgcagcttagcttcaagaacatgtgtaagctgcggcccttgctgcagaagtgggtggaggaagctgacaacaatgaaaatcttcaggagatatgcaaagcagaaaccctcgtgcaggcccgaaagagaaagcgaaccagtatcgagaaccgagtgagaggcaacctggagaatttgttcctgcagtgcccgaaacccacactgcagcagatcagccacatcgcccagcagcttgggctcgagaaggatgtggtccgagtgtggttctgtaaccggcgccagaagggcaagcgatcaagcagcgactatgcacaacgagaggattttgaggctgctgggtctcctttctcagggggaccagtgtcctttcctctggccccagggccccattttggtaccccaggctatgggagccctcacttcactgcactgtactcctcggtccctttccctgagggggaagcctttccccctgtctccgtcaccactctgggctctcccatgcattcaaactga; (SEQ ID NO: 37)NM_002701: atggcgggacacctggcttcggatttcgccttctcgccccctccaggtggtggaggtgatgggccaggggggccggagccgggctgggttgatcctcggacctggctaagcttccaaggccctcctggagggccaggaatcgggccgggggttgggccaggctctgaggtgtgggggattcccccatgccccccgccgtatgagttctgtggggggatggcgtactgtgggccccaggttggagtggggctagtgccccaaggcggcttggagacctctcagcctgagggcgaagcaggagtcggggtggagagcaactccgatggggcctccccggagccctgcaccgtcacccctggtgccgtgaagctggagaaggagaagctggagcaaaacccggaggagtcccaggacatcaaagctctgcagaaagaactcgagcaatttgccaagctcctgaagcagaagaggatcaccctgggatatacacaggccgatgtggggctcaccctgggggttctatttgggaaggtattcagccaaacgaccatctgccgctttgaggctctgcagcttagcttcaagaacatgtgtaagctgcggcccttgctgcagaagtgggtggaggaagctgacaacaatgaaaatcttcaggagatatgcaaagcagaaaccctcgtgcaggcccgaaagagaaagcgaaccagtatcgagaaccgagtgagaggcaacctggagaatttgttcctgcagtgcccgaaacccacactgcagcagatcagccacatcgcccagcagcttgggctcgagaaggatgtggtccgagtgtggttctgtaaccggcgccagaagggcaagcgatcaagcagcgactatgcacaacgagaggattttgaggctgctgggtctcctttctcagggggaccagtgtcctttcctctggccccagggccccattttggtaccccaggctatgggagccctcacttcactgcactgtactcctcggtccctttccctgagggggaagcctttccccctgtctccgtcaccactctgggctctcccatgcattcaaactga; NM 013633; EF032593; NM 131112; NM001114955. (SEQ ID NO: 38)SOX2: NM_003106: tgtacaacatgatggagacggagctgaagccgccgggcccgcagcaaacttcggggggcggcggcggcaactccaccgcggcggcggccggcggcaaccagaaaaacagcccggaccgcgtcaagcggcccatgaatgccttcatggtgtggtcccgcgggcagcggcgcaagatggcccaggagaaccccaagatgcacaactcggagatcagcaagcgcctgggcgccgagtggaaacttttgtcggagacggagaagcggccgttcatcgacgaggctaagcggctgcgagcgctgcacatgaaggagcacccggattataaataccggccccggcggaaaaccaagacgctcatgaagaaggataagtacacgctgcccggcgggctgctggcccccggcggcaatagcatggcgagcggggtcggggtgggcgccggcctgggcgcgggcgtgaaccagcgcatggacagttacgcgcacatgaacggctggagcaacggcagctacagcatgatgcaggaccagctgggctacccgcagcacccgggcctcaatgcgcacggcgcagcgcagatgcagcccatgcaccgctacgacgtgagcgccctgcagtacaactccatgaccagctcgcagacctacatgaacggctcgcccacctacagcatgtcctactcgcagcagggcacccctggcatggctcttggctccatgggttcggtggtcaagtccgaggccagctccagcccccctgtggttacctcttcctcccactccagggcgccctgccaggccggggacctccgggacatgatcagcatgtatctccccggcgccgaggtgccggaacccgccgcccccagcagacttcacatgtcccagcactaccagagcggcccggtgcccggcacggccattaacggcacactgcccctctcacacatgtga; NM 011443; NM 00110918. (SEQ ID NO: 39)FGF4: NM_002007:  atgtcggggcccgggacggccgcggtagcgctgctcccggcggtcctgctggccttgctggcgccctgggcgggccgagggggcgccgccgcacccactgcacccaacggcacgctggaggccgagctggagcgccgctgggagagcctggtggcgctctcgttggcgcgcctgccggtggcagcgcagcccaaggaggcggccgtccagagcggcgccggcgactacctgctgggcatcaagcggctgcggcggctctactgcaacgtgggcatcggcttccacctccaggcgctccccgacggccgcatcggcggcgcgcacgcggacacccgcgacagcctgctggagctctcgcccgtggagcggggcgtggtgagcatcttcggcgtggccagccggttcttcgtggccatgagcagcaagggcaagctctatggctcgcccttcttcaccgatgagtgcacgttcaaggagattctccttcccaacaactacaacgcctacgagtcctacaagtaccccggcatgttcatcgccctgagcaagaatgggaagaccaagaaggggaaccgagtgtcgcccaccatgaaggtcacccacttcctccccaggctgtga; NM 010202; NM 004380; NM 001025432; NM 004379.2;NP 004370; NM 134442; NP 604391. (SEQ ID NO: 40)Gata2: NM_032638: atggaggtggcgccggagcagccgcgctggatggcgcacccggccgtgctgaatgcgcagcaccccgactcacaccacccgggcctggcgcacaactacatggaacccgcgcagctgctgcctccagacgaggtggacgtcttcttcaatcacctcgactcgcagggcaacccctactatgccaaccccgctcacgcgcgggcgcgcgtctcctacagccccgcgcacgcccgcctgaccggaggccagatgtgccgcccacacttgttgcacagcccgggtttgccctggctggacgggggcaaagcagccctctctgccgctgcggcccaccaccacaacccctggaccgtgagccccttctccaagacgccactgcacccctcagctgctggaggccctggaggcccactctctgtgtacccaggggctgggggtgggagcgggggaggcagcgggagctcagtggcctccctcacccctacagcagcccactctggctcccaccttttcggcttcccacccacgccacccaaagaagtgtctcctgaccctagcaccacgggggctgcgtctccagcctcatcttccgcggggggtagtgcagcccgaggagaggacaaggacggcgtcaagtaccaggtgtcactgacggagagcatgaagatggaaagtggcagtcccctgcgcccaggcctagctactatgggcacccagcctgctacacaccaccccatccccacctacccctcctatgtgccggcggctgcccacgactacagcagcggactcttccaccccggaggcttcctggggggaccggcctccagcttcacccctaagcagcgcagcaaggctcgttcctgttcagaaggccgggagtgtgtcaactgtggggccacagccacccctctctggcggcgggacggcaccggccactacctgtgcaatgcctgtggcctctaccacaagatgaatgggcagaaccgaccactcatcaagcccaagcgaagactgtcggccgccagaagagccggcacctgttgtgcaaattgtcagacgacaaccaccaccttatggcgccgaaacgccaacggggaccctgtctgcaacgcctgtggcctctactacaagctgcacaatgttaacaggccactgaccatgaagaaggaagggatccagactcggaaccggaagatgtccaacaagtccaagaagagcaagaaaggggcggagtgcttcgaggagctgtcaaagtgcatgcaggagaagtcatcccccttcagtgcagctgccctggctggacacatggcacctgtgggccacctcccgcccttcagccactccggacacatcctgcccactccgacgcccatccacccctcctccagcctctccttcggccacccccacccgtccagcatggtgaccgccatgggctag; AB 102789; AB 102790; NM 008090.(SEQ ID NO: 41) Gata3: NM 001002295:atggaggtgacggcggaccagccgcgctgggtgagccaccaccaccccgccgtgctcaacgggcagcacccggacacgcaccacccgggcctcagccactcctacatggacgcggcgcagtacccgctgccggaggaggtggatgtgctttttaacatcgacggtcaaggcaaccacgtcccgccctactacggaaactcggtcagggccacggtgcagaggtaccctccgacccaccacgggagccaggtgtgccgcccgcctctgcttcatggatccctaccctggctggacggcggcaaagccctgggcagccaccacaccgcctccccctggaatctcagccccttctccaagacgtccatccaccacggctccccggggcccctctccgtctaccccccggcctcgtcctcctccttgtcggggggccacgccagcccgcacctcttcaccttcccgcccaccccgccgaaggacgtctccccggacccatcgctgtccaccccaggctcggccggctcggcccggcaggacgagaaagagtgcctcaagtaccaggtgcccctgcccgacagcatgaagctggagtcgtcccactcccgtggcagcatgaccgccctgggtggagcctcctcgtcgacccaccaccccatcaccacctacccgccctacgtgcccgagtacagctccggactcttcccccccagcagcctgctgggcggctcccccaccggcttcggatgcaagtccaggcccaaggcccggtccagcacagaaggcagggagtgtgtgaactgtggggcaacctcgaccccactgtggcggcgagatggcacgggacactacctgtgcaacgcctgcgggctctatcacaaaatgaacggacagaaccggcccctcattaagcccaagcgaaggctgtctgcagccaggagagcagggacgtcctgtgcgaactgtcagaccaccacaaccacactctggaggaggaatgccaatggggaccctgtctgcaatgcctgtgggctctactacaagcttcacaatattaacagacccctgactatgaagaaggaaggcatccagaccagaaaccgaaaaatgtctagcaaatccaaaaagtgcaaaaaagtgcatgactcactggaggacttccccaagaacagctcgtttaacccggccgccctctccagacacatgtcctccctgagccacatctcgcccttcagccactccagccacatgctgaccacgcccacgccgatgcacccgccatccagcctgtcctttggaccacaccacccctccagcatggtcaccgccatgggttag; NM 008091; AM392688; AM392571; NM 001002295; NM 002051. (SEQ ID NO: 42)Gata4: BC101580:atgtatcagagcttggccatggccgccaaccacgggccgccccccggtgcctacgaggcgggcggccccggcgccttcatgcacggcgcgggcgccgcgtcctcgccagtctacgtgcccacaccgcgggtgccctcctccgtgctgggcctgtcctacctccagggcggaggcgcgggctctgcgtccggaggcgcctcgggcggcagctccggtggggccgcgtctggtgcggggcccgggacccagcagggcagcccgggatggagccaggcgggagccgacggagccgcttacaccccgccgccggtgtcgccgcgcttctccttcccggggaccaccgggtccctggcggccgccgccgccgctgccgcggcccgggaagctgcggcctacagcagtggcggcggagcggcgggtgcgggcctggcgggccgcgagcagtacgggcgcgccggcttcgcgggctcctactccagcccctacccggcttacatggccgacgtgggcgcgtcctgggccgcagccgccgccgcctccgccggccccttcgacagcccggtcctgcacagcctgcccggccgggccaacccggccgcccgacaccccaatctcgatatgtttgacgacttctcagaaggcagagagtgtgtcaactgtggggctatgtccaccccgctctggaggcgagatgggacgggtcactatctgtgcaacgcctgcggcctctaccacaagatgaacggcatcaaccggccgctcatcaagcctcagcgccggctgtccgcctcccgccgagtgggcctctcctgtgccaactgccagaccaccaccaccacgctgtggcgccgcaatgcggagggcgagcctgtgtgcaatgcctgcggcctctacatgaagctccacggggtccccaggcctcttgcaatgcggaaagaggggatccaaaccagaaaacggaagcccaagaacctgaataaatctaagacaccagcagctccttcaggcagtgagagccttcctcccgccagcggtgcttccagcaactccagcaacgccaccaccagcagcagcgaggagatgcgtcccatcaagacggagcctggcctgtcatctcactacgggcacagcagctccgtgtcccagacgttctcagtcagtgcgatgtctggccatgggccctccatccaccctgtcctctcggccctgaagctctccccacaaggctatgcgtctcccgtcagccagtctccacagaccagctccaagcaggactcttggaacagcctggtcttggccgacagtcacggggacataatcactgcgtaa;AF179424; DQ666280. (SEQ ID NO: 43) Gata5: BC117356:atgtaccagagcctggcgctggccgcgagcccccgccaggccgcctacgccgactcgggctccttcctgcacgctccgggcgccggctctccgatgtttgtgccgccggcgcgcgtcccctcgatgctgtcctacctgtccgggtgtgagccgagcccgcagccccccgagctcgctgcgcgccccggctgggcgcagacagccaccgcggattcgtcggccttcggcccgggcagtccgcaccccccagccgcgcacccgcccggggccaccgccttccctttcgcgcacagcccctcggggcccggcagcggcggcagcgcggggggccgagacggcagtgcctaccagggcgcgctgttgcctcgagaacagttcgcggccccgcttgggcggccggtggggacctcgtactccgccacctacccggcctacgtgagccccgacgtggcccagtcctggactgccgggcccttcgatggcagcgtcctgcacggcctcccaggccgcaggcccaccttcgtgtccgacttcttggaggagttcccgggtgagggtcgtgagtgtgtcaactgcggggccctgtccacaccgctgtggcgccgagatggcaccggccactacctgtgcaatgcctgcggcctctaccacaagatgaatggcgtcaaccggccgctcgttcggcctcagaagcgcctgtcctcgtcccgccgcgccggcctctgctgcaccaactgccacacgaccaacaccacgctgtggcggcggaactcggagggggagcccgtgtgcaatgcctgcggcctctacatgaagctgcacggggtgccgcggcctctggctatgaagaaagaaagcatccagacacggaagcggaagccaaagaccatcgccaaggccaggggctcctcaggatccacaaggaatgcctcggcctccccatctgctgtcgccagcactgacagctcagcagccacttccaaagccaagcccagcctggcgtccccagtgtgccctgggcccagcatggccccccaggcctctggccaggaggatgactctcttgcccccggccacttggagttcaagttcgagcctgaggactttgccttcccctccacggccccgagcccccaggctggcctcaggggggctctgcgccaagaggcctggtgtgcgctggccttggcctag; BC 105654. (SEQ ID NO: 44)Gata6: NM_005257: atggccttgactgacggcggctggtgcttgccgaagcgcttcggggccgcgggtgcggacgccagcgactccagagcctttccagcgcgggagccctccacgccgccttcccccatctcttcctcgtcctcctcctgctcccggggcggagagcggggccccggcggcgccagcaactgcgggacgcctcagctcgacacggaggcggcggccggacccccggcccgctcgctgctgctcagttcctacgcttcgcatcccttcggggctccccacggaccttcggcgcctggggtcgcgggccccgggggcaacctgtcgagctgggaggacttgctgctgttcactgacctcgaccaagccgcgaccgccagcaagctgctgtggtccagccgcggcgccaagctgagccccttcgcacccgagcagccggaggagatgtaccagaccctcgccgctctctccagccagggtccggccgcctacgacggcgcgcccggcggcttcgtgcactctgcggccgcggcggcagcagccgcggcggcggccagctccccggtctacgtgcccaccacccgcgtgggttccatgctgcccggcctaccgtaccacctgcaggggtcgggcagtgggccagccaaccacgcgggcggcgcgggcgcgcaccccggctggcctcaggcctcggccgacagccctccatacggcagcggaggcggcgcggctggcggcggggccgcggggcctggcggcgctggctcagccgcggcgcacgtctcggcgcgcttcccctactctcccagcccgcccatggccaacggcgccgcgcgggagccgggaggctacgcggcggcgggcagtgggggcgcgggaggcgtgagcggcggcggcagtagcctggcggccatgggcggccgcgagccccagtacagctcgctgtcggccgcgcggccgctgaacgggacgtaccaccaccaccaccaccaccaccaccaccatccgagcccctactcgccctacgtgggggcgccactgacgcctgcctggcccgccggacccttcgagaccccggtgctgcacagcctgcagagccgcgccggagccccgctcccggtgccccggggtcccagtgcagacctgctggaggacctgtccgagagccgcgagtgcgtgaactgcggctccatccagacgccgctgtggcggcgggacggcaccggccactacctgtgcaacgcctgcgggctctacagcaagatgaacggcctcagccggcccctcatcaagccgcagaagcgcgtgccttcatcacggcggcttggattgtcctgtgccaactgtcacaccacaactaccaccttatggcgcagaaacgccgagggtgaacccgtgtgcaatgcttgtggactctacatgaaactccatggggtgcccagaccacttgctatgaaaaaagagggaattcaaaccaggaaacgaaaacctaagaacataaataaatcaaagacttgctctggtaatagcaataattccattcccatgactccaacttccacctcttctaactcagatgattgcagcaaaaatacttcccccacaacacaacctacagcctcaggggcgggtgccccggtgatgactggtgcgggagagagcaccaatcccgagaacagcgagctcaagtattcgggtcaagatgggctctacataggcgtcagtctcgcctcgccggccgaagtcacgtcctccgtgcgaccggattcctggtgcgccctggccctggcctga;AF179425; EF444980; NM 005257; NP 005248. (SEQ ID NO: 45)HNF1: NM_000458: atggtgtccaagctcacgtcgctccagcaagaactcctgagcgccctgctgagctccggggtcaccaaggaggtgctggttcaggccttggaggagttgctgccatccccgaacttcggggtgaagctggagacgctgcccctgtcccctggcagcggggccgagcccgacaccaagccggtcttccatactctcaccaacggccacgccaagggccgcttgtccggcgacgagggctccgaggacggcgacgactatgacacacctcccatcctcaaggagctgcaggcgctcaacaccgaggaggcggcggagcagcgggcggaggtggaccggatgctcagtgaggacccttggagggctgctaaaatgatcaagggttacatgcagcaacacaacatcccccagagggaggtggtcgatgtcaccggcctgaaccagtcgcacctctcccagcatctcaacaagggcacccctatgaagacccagaagcgtgccgctctgtacacctggtacgtcagaaagcaacgagagatcctccgacaattcaaccagacagtccagagttctggaaatatgacagacaaaagcagtcaggatcagctgctgtttctctttccagagttcagtcaacagagccatgggcctgggcagtccgatgatgcctgctctgagcccaccaacaagaagatgcgccgcaaccggttcaaatgggggcccgcgtcccagcaaatcttgtaccaggcctacgatcggcaaaagaaccccagcaaggaagagagagaggccttagtggaggaatgcaacagggcagaatgtttgcagcgaggggtgtccccctccaaagcccacggcctgggctccaacttggtcactgaggtccgtgtctacaactggtttgcaaaccgcaggaaggaggaggcattccggcaaaagctggccatggacgcctatagctccaaccagactcacagcctgaaccctctgctctcccacggctccccccaccaccagcccagctcctctcctccaaacaagctgtcaggagtgcgctacagccagcagggaaacaatgagatcacttcctcctcaacaatcagtcaccatggcaacagcgccatggtgaccagccagtcggttttacagcaagtctccccagccagcctggacccaggccacaatctcctctcacctgatggtaaaatgatctcagtctcaggaggaggtttgcccccagtcagcaccttgacgaatatccacagcctctcccaccataatccccagcaatctcaaaacctcatcatgacacccctctctggagtcatggcaattgcacaaagcctcaacacctcccaagcacagagtgtccctgtcatcaacagtgtggccggcagcctggcagccctgcagcccgtccagttctcccagcagctgcacagccctcaccagcagcccctcatgcagcagagcccaggcagccacatggcccagcagcccttcatggcagctgtgactcagctgcagaactcacacatgtacgcacacaagcaggaacccccccagtattcccacacctcccggtttccatctgcaatggtggtcacagataccagcagcatcagtacactcaccaacatgtcttcaagtaaacagtgtcctctacaagcctggtga;NM_013103; NM 000545; NM 009327; (SEQ ID NO: 46) NM 012669:atggtttctaagttgagccagctgcagacggagctcctggctgctctgctcgagtcgggcctgagcaaagaggctctgatccaggctctgggggagcccgggccctacctgatggttggagatggtcccctggacaagggggagtcctgcggtgggactcgaggggacctgaccgagctgcccaatggcctgggggagacgcgtggctcggaagatgacacggatgacgatggggaagacttcgcgccacccattctgaaagagctggagaacctcagcccagaggaggcagcccaccagaaagccgtggtggagtcacttcttcaggaggacccatggcgcgtggcaaagatggtcaagtcgtacctgcagcaacacaacatcccccagcgggaggtggtggacactacgggtctcaaccagtcccacctgtcccagcacctcaacaagggcacccccatgaagacgcagaagcgggccgcgctgtacacctggtacgtccgcaagcagcgagaggtggctcagcaattcacccacgcggggcagggcggactgattgaagagcccacaggtgatgagctgccaaccaaaaaggggcggaggaaccggttcaagtggggccccgcatcccagcagatcctgttccaggcttacgagaggcagaagaaccccagcaaggaagagcgagagaccttggtggaggagtgcaatagggcggagtgcatccagagaggggtgtcaccatcgcaggcccaggggctaggctccaaccttgtcaccgaggtgcgtgtctacaactggtttgccaaccggcgcaaggaagaagcctttcggcataagctggccatggacacgtataacgggcctccacccgggccaggccccggccctgcgctacctgcccacagttccccgggcctgcccacaaccaccctctctcccagtaaggtccacggtgtgcggtatggacagtctgcaaccagcgaggcagctgaggtgccctccagcagcggaggtcccttagtcacagtgtctgcggccttacaccaagtgtcccccacaggcttggagcccagcagcctgctgagcaccgaggccaagctggtctcagccacggggggtcccctgcctcccgtcagcaccctgacagcactgcacagcttggagcagacgtctccaggtctcaaccagcagccgcagaaccttatcatggcctcgctgcctggggtcatgaccatcggcccaggggagcccgcctccctgggtcccacgttcactaacacgggtgcctctaccctggtcattggtctggcctccacacaggcacagagcgtgccagtcatcaacagcatggggagcagcctgaccaccctgcagccggtccagttttcccagccactgcacccttcctatcagcagcctctcatgccccctgtacagagccacgtggcccagagtcccttcatggcaaccatggcccagctgcagagcccccacgccctgtacagccacaagcctgaggtggcccagtacacgcatacaagcctgcttccgcagaccatgctgatcacagacaccaacctcagcacccttgccagcctcacgcccaccaagcaggtcttcacctcagacacagaggcctccagtgagcctgggcttcatgagccgtcgtctccagccacaaccattcacatccccagccaggacccgtcaaacatccagcacctgcagcctgctcaccggctcagcaccagtcccacagtgtcctccagcagcctggtgttgtaccagagttctgactccaacgggcacagccacctgctgccatccaaccacggtgtcatcgagacttttatctccacccagatggcctcctcctcccagtaa; NM_009330.(SEQ ID NO: 47)HNF3: X74936: atgttagggactgtgaagatggaagggcatgagagcaacgactggaacagctactacgcggacacgcaggaggcctactcctctgtccctgtcagcaacatgaactccggcctgggctctatgaactccatgaacacctacatgaccatgaacaccatgaccacgagcggcaacatgaccccggcttccttcaacatgtcctacgccaacacgggcttaggggccggcctgagtcccggtgctgtggctggcatgccaggggcctctgcaggcgccatgaacagcatgactgcggcgggcgtcacggccatgggtacggcgctgagcccgggaggcatgggctccatgggcgcgcagcccgtcacctccatgaacggcctgggtccctacgccgccgccatgaacccgtgcatgagtcccatggcgtacgcgccgtccaacctgggccgcagccgcgcggggggcggcggcgacgccaagacattcaagcgcagctaccctcacgccaagccgccttactcctacatctcgctcatcacgatggccatccagcaggcgcccagcaagatgctcacgctgagcgagatctaccagtggatcatggacctcttcccctattaccgccagaaccagcagcgctggcagaactccatccgccactcgctgtccttcaacgattgtttcgtcaaggtggcacgatccccagacaagccaggcaagggctcctactggacgctgcacccggactccggcaacatgttcgagaacggctgctacttgcgccgccaaaagcgcttcaagtgtgagaagcagccgggggccggaggtgggagtgggggcggcggctccaaagggggcccagaaagtcgcaaggacccctcaggcccggggaaccccagcgccgagtcaccccttcattggggtgtgcacggaaaggctagccagctagagggcgcgccggcccccgggcccgccgccagcccccagactctggaccacagcggggccacggcgacagggggcgcttcggagttgaagtctccagcgtcttcatctgcgccccccataagctccgggccaggggcgctggcatctgtacccccctctcacccggctcacggcctggcaccccacgaatctcagctgcatctgaaaggggatccccactactcctttaatcaccccttctccatcaacaacctcatgtcctcctccgagcaacagcacaagctggacttcaaggcatacgagcaggcgctgcagtactctccttatggcgctaccttgcccgccagtctgccccttggcagcgcctcagtggccacgaggagccccatcgagccctcagccctggagccagcctactaccaaggtgtgtattccagacccgtgctaaatacttcctag;(SEQ ID NO: 48)HNF3gammaX74938M: atgctgggctcagtgaagatggaggctcatgacctggccgagtggagctactacccggaggcgggcgaggtgtattctccagtgaatcctgtgcccaccatggcccctctcaactcctacatgaccttgaacccactcagctctccctaccctcccggagggcttcaggcctccccactgcctacaggacccctggcacccccagcccccactgcgcccttggggcccaccttcccaagcttgggcactggtggcagcaccggaggcagtgcttccgggtatgtagccccagggcccgggcttgtacatggaaaagagatggcaaaggggtaccggcggccactggcccacgccaaaccaccatattcctacatctctctcataaccatggctattcagcaggctccaggcaagatgctgaccctgagtgaaatctaccaatggatcatggacctcttcccgtactaccgggagaaccagcaacgttggcagaactccatccggcattcgctgtccttcaatgactgcttcgtcaaggtggcacgctccccagacaagccaggcaaaggctcctactgggccttgcatcccagctctgggaacatgtttgagaacggatgctatctccgccggcagaagcgcttcaagctggaggagaaggcaaagaaaggaaacagcgccacatcggccagcaggaatggtactgcggggtcagccacctctgccaccactacagctgccactgcagtcacctccccggctcagccccagcctacgccatctgagcccgaggcccagagtggggatgatgtggggggtctggactgcgcctcacctccttcgtccacaccttatttcagcggcctggagctcccgggggaactaaagttggatgcgccctataacttcaaccaccctttctctatcaacaacctgatgtcagaacagacatcgacaccttccaaactggatgtggggtttgggggctacggggctgagagtggggagcctggagtctactaccagagcctctattcccgctctctgcttaatgcatcctag; (SEQ ID NO: 49)HNF3betaX74937: atgctgggagccgtgaagatggaagggctcgagccatccgactggagcagctactacgcggagcccgagggctactcttccgtgagcaacatgaacgccggcctggggatgaatggcatgaacacatacatgagcatgtccgcggctgccatgggcggcggttccggcaacatgagcgcgggctccatgaacatgtcatcctatgtgggcgctggaatgagcccgtcgctagctggcatgtccccgggcgccggcgccatggcgggcatgagcggctcagccggggcggccggcgtggcgggcatgggacctcacctgagtccgagtctgagcccgctcgggggacaggcggccggggccatgggtggccttgccccctacgccaacatgaactcgatgagccccatgtacgggcaggccggcctgagccgcgctcgggaccccaagacataccgacgcagctacacacacgccaaacctccctactcgtacatctcgctcatcaccatggccatccagcagagccccaacaagatgctgacgctgagcgagatctatcagtggatcatggacctcttccctttctaccggcagaaccagcagcgctggcagaactccatccgccactctctctccttcaacgactgctttctcaaggtgccccgctcgccagacaagcctggcaagggctccttctggaccctgcacccagactcgggcaacatgttcgagaacggctgctacctgcgccgccagaagcgcttcaagtgtgagaagcaactggcactgaaggaagccgcgggtgcggccagtagcggaggcaagaagaccgctcctgggtcccaggcctctcaggctcagctcggggaggccgcgggctcggcctccgagactccggcgggcaccgagtccccccattccagcgcttctccgtgtcaggagcacaagcgaggtggcctaagcgagctaaagggagcacctgcctctgcgctgagtcctcccgagccggcgccctcgcctgggcagcagcagcaggctgcagcccacctgctgggcccacctcaccacccaggcctgccaccagaggcccacctgaagcccgagcaccattacgccttcaaccaccccttctctatcaacaacctcatgtcgtccgagcagcaacatcaccacagccaccaccaccatcagccccacaaaatggacctcaaggcctacgaacaggtcatgcactacccagggggctatggttcccccatgccaggcagcttggccatgggcccagtcacgaacaaagcgggcctggatgcctcgcccctggctgcagacacttcctactaccaaggagtgtactccaggcctattatgaactcatcctaa; (SEQ ID NO: 50)HNF3G: AH008133: atgctgggctcagtgaagatggaggcccatgacctggccgagtggagctactacccggaggcgggcgaggtctactcgccggtgaccccagtgcccaccatggcccccctcaactcctacatgaccctgaatcctctaagctctccctatccccctggggggctccctgcctccccactgccctcaggacccctggcacccccagcacctgcagcccccctggggcccactttcccaggcctgggtgtcagcggtggcagcagcagctccgggtacggggccccgggtcctgggctggtgcacgggaaggagatgccgaaggggtatcggcggcccctggcacacgccaagccaccgtattcctatatctcactcatcaccatggccatccagcaggcgccgggcaagatgctgaccttgagtgaaatctaccagtggatcatggacctcttcccttactaccgggagaatcagcagcgctggcagaactccattcgccactcgctgtctttcaacgactgcttcgtcaaggtggcgcgttccccagacaagcctggcaagggctcctactgggccctacaccccagctcagggaacatgtttgagaatggctgctacctgcgccgccagaaacgcttcaagctggaggagaaggtgaaaaaagggggcagcggggctgccaccaccaccaggaacgggacagggtctgctgcctcgaccaccacccccgcggccacagtcacctccccgccccagcccccgcctccagcccctgagcctgaggcccagggcggggaagatgtgggggctctggactgtggctcacccgcttcctccacaccctatttcactggcctggagctcccaggggagctgaagctggacgcgccctacaacttcaaccaccctttctccatcaacaacctaatgtcagaacagacaccagcacctcccaaactggacgtggggtttgggggctacggggctgaaggtggggagcctggagtctactaccagggcctctattcccgctctttgcttaatgcatcctag; (SEQ ID NO: 51)HNF3A: AH008132: atgttaggaactgtgaagatggaagggcatgaaaccagcgactggaacagctactacgcagacacgcaggaggcctactcctcggtcccggtcagcaacatgaactcaggcctgggctccatgaactccatgaacacctacatgaccatgaacaccatgactacgagcggcaacatgaccccggcgtccttcaacatgtcctatgccaacccggccttaggggccggcctgagtcccggcgcagtagccggcatgccggggggctcggcgggcgccatgaacagcatgactgcggccggcgtgacggccatgggtacggcgctgagcccgagcggcatgggcgccatgggtgcgcagcaggcggcctccatgatgaatggcctgggcccctacgcggccgccatgaacccgtgcatgagccccatggcgtacgcgccgtccaacctgggccgcagccgcgcgggcggcggcggcgacgccaagacgttcaagcgcagttacccgcacgccaagccgccctactcgtacatctcgctcatcaccatggccatccagcgggcgcccagcaagatgctcacgctgagcgagatctaccagtggatcatggacctcttcccctattaccggcagaaccagcagcgctggcagaactccatccgccactcgctgtccttcaatgactgcttcgtcaaggtggcacgctccccggacaagccgggcaagggctcctactggacgctgcacccggactccggcaacatgttcgagaacggctgctacttgcgccgccagaagcgcttcaagtgcgagaagcagccgggggccggcggcgggggcgggagcggaagcgggggcagcggcgccaagggcggccctgagagccgcaaggacccctctggcgcctctaaccccagcgccgactcgcccctccatcggggtgtgcacgggaagaccggccagctagagggcgcgccggccccgggcccggccgccagcccccagactctggaccacagtggggcgacggcgacagggggcgcctcggagttgaagactccagcctcctcaactgcgccccccataagctccgggcccggggcgctggcctctgtgcccgcctctcacccggcacacggcttggcaccccacgagtcccagctgcacctgaaaggggacccccactactccttcaaccacccgttctccatcaacaacctcatgtcctcctcggagcagcagcataagctggacttcaaggcatacgaacaggcactgcaatactcgccttacggctctacgttgcccgccagcctgcctctaggcagcgcctcggtgaccaccaggagccccatcgagccctcagccctggagccggcgtactaccaaggtgtgtattccagacccgtcctaaacacttcctag; (SEQ ID NO: 52)HNF4alpha: NM_008261: atgcgactctctaaaacccttgccggcatggatatggccgactacagcgctgccctggacccagcctacaccaccctggagtttgaaaatgtgcaggtgttgaccatgggcaatgacacgtccccatctgaaggtgccaacctcaattcatccaacagcctgggcgtcagtgccctgtgcgccatclgtggcgaccgggccaccggcaaacactacggagcctcgagctgtgacggctgcaaggggttcttcaggaggagcgtgaggaagaaccacatgtactcctgcaggtttagccgacaalgtgtggtagacaaagataagaggaaccagtgtcgttactgcaggcttaagaagtgcttccgggctggcatgaagaaggaagclgtccaaaatgagcgggaccggatcagcacgcggaggtcaagctacgaggacagcagcctgccctccatcaacgcgctcclgcaggcagaggttctgtcccagcagatcacctctcccalctclgggatcaatggcgacattcgggcaaagaagattgccaacatcacagacgtgtgtgagtctatgaaggagcagctgctggtcctggtcgagtgggccaagtacalcccggccttctgcgaactccttctggatgaccaggtggcgctgctcagggcccacgccggtgagcatctgctgcttggagccaccaagaggtccatggtgtttaaggacgtgctgctcctaggcaatgactacatcgtccctcggcactgtccagagctagcggagatgagccgtgtgtccatccgcatcctcgatgagctggtcctgccctlccaagagctgcagattgatgacaatgaatatgcctgcctcaaagccalcatcttctttgatccagatgccaaggggctgagtgacccgggcaagatcaagcggctgcggtcacaggtgcaagtgagcctggaggattacalcaacgaccggcagtacgactctcggggccgctttggagagctgctgctgctgttgcccacgctgcagagcatcacctggcagatgatcgaacagatccagttcatcaagctcttcggcatggccaagattgacaacctgctgcaggagatgcttctcggaggglctgccagtgatgcaccccacacccaccaccccctgcaccctcacctgatgcaagaacacatgggcaccaatgtcattgttgctaacacgatgccctctcacctcagcaatggacagatgtgtgaglggccccgacccagggggcaggcagccactcccgagactccacagccatcaccaccaagtggctcgggatctgaatcclacaagctcctgccaggagccatcaccaccatcgtcaagcclccctctgccattccccagccaacgatcaccaagcaagaagccatctag;(SEQ ID NO: 53)I1NF4a: NM022180: atggacatggctgactacagtgctgccttggacccagcclacaccaccctggagtttgaaaatgtgcaggtgttgaccatgggcaatgacacatccccatctgaaggtgccaacctcaactcatccaacagcctgggtgtcagtgccctgtgtgccatctgtggcgatcgggccactggcaaacactacggagcctcaagctglgacggclgcaagggattcltcaggaggagcgtgaggaagaaccacatgtactcctgcaggtttagcaggcagtgcgtggtagacaaagalaagaggaaccagtgtcgttactgcaggctcaagaagtgcttccgggctggcatgaagaaagaagccgtccaaaatgagcgggatcggatcagcacgcggaggtcaagctacgaggacagcagcctaccctccattaatgcgctcctgcaggcagaggtcctgtctcagcagatcacctcccccatctctgggatcaatggcgacattcgggccaagaagattgccaacatcacggatglgtgtgagtctatgaaggagcagctgctggttctgglcgaatgggccaagtacatcccggccttctgtgaacttcttctggatgaccaggtggcgclgclcagagcccacgctggtgagcacctgctgctlggagccaccaagaggtccatggtgttcaaggatglgctgctcclaggcaatgactacatcgtccctcggcactgtccagagctagcagagatgagccgtgtgtccattcgcatcctcgatgagctggtcttgcccttccaagagctgcagalcgatgataatgaatacgcctgcctcaaagccatcatcttctltgacccagatgccaaggggctgagtgacccaggcaagatcaagcggctgcggtcacaggtgcaggtgagcctggaggattacatcaacgaccggcagtatgactctcggggtcgttltggagagctgctgctgctcctgcccactctgcagagcattacclggcagatgatcgagcagatccagttcatcaagctctttggcatggccaagattgacaacctgctgcaggagatgctgcttggagggtctgccagtgacgcgccccacgcccaccaccccctgcaccctcacctgatgcaagaacacatgggcaccaatgtcatagttgccaacacgatgccctctcacctcagcaatggacagatgtgtgagtggccccggcccagggggcaggcagccacccctgagactccacagccatcaccaccaagtggctctggatctgaatcctacaagctcctgccaggagccatcaccaccatcgtcaagcctccctctgccatcccccagccaacgatcaccaagcaggaagccatclag; (SEQ ID NO: 54)HNF6: U95945: atgaacgcacagctgaccatggaggcgatcggcgagctgcacggggtgagccatgagccggtgcccgcccctgctgacctgctgggcggcagccctcacgcgcgcagctccgtgggacaccgcggcagccacctgcctcccgcgcacccgcgttccatgggcatggcgtccctgctggacggcggcagcggaggcagcgattaccaccaccaccaccgcgcccctgagcacagcttggctggccccctgcaccccaccatgaccatggcctgtgaaactcccccaggtatgagcatgcccaccacctacactaccttaacccclclgcagccgclgccgcccatctccaccgtgtccgacaagttccctcaccatcatcaccaccaccatcaccaccaccacccacaccaccaccagcgcctggcgggcaacgtgagcggtagtttcacacttatgcgggatgagcgcgggctggcctctatgaataacctctataccccctaccacaaggacgtggctggcatgggccagagcctctcgcccctctctggctccggtctgggcagcattcacaactcccagcaaggacttccccactatgctcatcccggcgcggctatgcccaccgacaagatgctcaccccaaatggctttgaagcccaccaccctgccatgctcggtcgccacggggagcagcacctcacgcccacctcggccggcatggtacccatcaacggccttcctccgcaccatcctcatgcccacctgaatgcccagggccacggacagctcctgggcacagcccgagagcccaacccttcggtgaccggcgcgcaggtcagcaatggaagtaattcagggcagatggaagagatcaataccaaagaggtggcgcagcgtatcaccaccgagctcaaacgttacagcatcccacaggccatcttcgcgcagagggtgctctgccgttcccaggggaccctttcggacctgctgcgaaaccccaagccctggagcaaactcaagtcgggtcgggagaccttccggaggatgtggaagtggctgcaggagccggagttccagcgcatgtcggcgctccgcttagcagcctgcaaacggaaagagcaagaacatgggaaggacagaggcaacacccccaaaaagcccaggctggtcttcacagacgtccaacgtcgaactctacatgcaatattcaaggaaaataagcgtccgtccaaagaattacaaatcaccatctcccagcagctggggttggagctgagcactgtcagcaacttcttcatgaatgccagaaggaggagtctggacaagtggcaggacgagggcggctccaactcaggcagttcatcgtcctcatcgagcacttgtaccaaagcatga; (SEQ ID NO: 55)IILXB9: NM001096823:atggagaagtccaagaatttcaggattgacgctctcctggcgatagatccccccaaggctcagacctccccattggctctggtcacctcgctgtcctcctcgtctctctccgggagccccccgtccgagcacactgacagcctcaggactgactccccctcccctccaaggacttgtggactggtccctaaaccaggtttcctgagcagccaccagcaccccccaaacatgatgtcattgcacccccaggctgctccagggatcccccctcaggccctgtatggacacccgatgtacagctacttggcagcggggcagcacccagctctgtcctacccctactcccagatgcagagcagccaccacccccaccccatggaccccatcaagatcagcgctggcaccttccaactggaccagtggctcagagcctccactgccggcatgatgctgcccaaaatggcagactttaactcccaggcccaatccaacctgctgggaaagtgcagaagaccaaggacagcgtttaccagtcagcagctgttggaactggagcaccaattcaagctgaacaagtacctctccaggccgaaacgctttgaagtggccacttccctgatgctcactgagacgcaggtgaagatctggttccagaacaggcgcatgaaatggaagaggagtaagaaagccaaggagcaggcggcgcaggactcagcagagaaacagcagagggcaggcaagggcagcagcgaggagaagtgctcggatgagctgcaggaagagaagaaatcctaccatctccatcccaggggggagcccatcaaagggaacggccgcctgcagcccagagactatacagacagcgaagaggacgaggaggaggacagggaagaggaggaagaggaagatcacagaggggaggggaagcggttttaccatcattcttctgactgcacatccgaggaagaggagaacagccacaataagcagagcggccactga; (SEQ ID NO: 56) NM 019944.atggaaaaatccaaaaatttccgcatcgacgccctgctggccgtggatcccccgcgagccgcctccacgcagagcgcgcctctggccttggtcacttccctcgcgactacagtatctggtcccggccgcggcggcagcggcggcggggggaccagtagcggggcgagccgtagctgcagtcccgcatcctcggaggccactgcagcgcccggtgaccggctgagagctgagagcccgtcgcccccacgcttgctggctgcacactgcgcgctgctgcccaagcccggattcctgggcgccggaggaggcggcggcgcggcgggtgggccgggcactccccaccaccacgcgcaccctggtgcagcagccgccgcggctgccgctgccgctgccgcggctgccggtggcctggcactggggctgcacccggggggcgcacagggcggcgcgggcctccctgcacaggcggctctctatggacacccggtctacagttattcggcagcagctgcagcggccgcgctagctggccagcacccggcgctttcctactcataccctcaggtgcagggcgcgcaccctgcgcaccctgccgaccccatcaagctgggtgccagcaccttccaactggaccagtggctgcgcgcgtctactgcgggcatgatcctgcccaagatgccggacttcagctgtcaggcgcagtcgaacctcttggggaagtgccgaaggcctcgcacggccttcaccagccagcagctgttggagctggaacaccagttcaagctcaacaagtacctgtctcgacccaagcgttttgaggtggctacctcgctcatgctcaccgagactcaggtgaagatttggttccagaaccgccgaatgaaatggaaacgcagcaaaaaggccaaagagcaggctgcgcaggaggcggagaagcagaagggcggcggcgggggcaccggcaaaggcggcagtgaggagaagacggaagaggagctgatggggcctccggtttcgggggacaaggcaagcggccgtcgcctgcgggacttgcgggacagtgaccctgatgaggacgaggatgatgaagaagaggacaacttcccgtacagcaatggtgccggtgcccatgctgcctcatccgactgctcatctgaggacgactcgcctcctccaagactaggcgggcctggacaccaacctctgccccagtag; (SEQ ID NO: 57)NM_005515: atggaaaaatccaaaaatttccgcatcgacgccctgctggccgtggatcccccgcgagccgcctccacgcagagcgcgcctctggccttggtcacttccctcgcgactacagtatctggtcccggccgcggcggcagcggcggcggggggaccagtagcggggcgagccgtagctgcagtcccgcatcctcggaggccactgcagcgcccggtgaccggctgagagctgagagcccgtcgcccccacgcttgctggctgcacactgcgcgctgctgcccaagcccggattcctgggcgccggaggaggcggcggcgcggcgggtgggccgggcactccccaccaccacgcgcaccctggtgcagcagccgccgcggctgccgctgccgctgccgcggctgccggtggcctggcactggggctgcacccggggggcgcacagggcggcgcgggcctccctgcacaggcggctctctatggacacccggtctacagttattcggcagcagctgcagcggccgcgctagctggccagcacccggcgctttcctactcataccctcaggtgcagggcgcgcaccctgcgcaccctgccgaccccatcaagctgggtgccagcaccttccaactggaccagtggctgcgcgcgtctactgcgggcatgatcctgcccaagatgccggacttcagctgtcaggcgcagtcgaacctcttggggaagtgccgaaggcctcgcacggccttcaccagccagcagctgttggagctggaacaccagttcaagctcaacaagtacctgtctcgacccaagcgttttgaggtggctacctcgctcatgctcaccgagactcaggtgaagatttggttccagaaccgccgaatgaaatggaaacgcagcaaaaaggccaaagagcaggctgcgcaggaggcggagaagcagaagggcggcggcgggggcaccggcaaaggcggcagtgaggagaagacggaagaggagctgatggggcctccggtttcgggggacaaggcaagcggccgtcgcctgcgggacttgcgggacagtgaccctgatgaggacgaggatgatgaagaagaggacaacttcccgtacagcaatggtgccggtgcccatgctgcctcatccgactgctcatctgaggacgactcgcctcctccaagactaggcgggcctggacaccaacctctgcc; (SEQ ID NO: 58) Lbx1: NM_006562:atgacttccaaggaggacggcaaggcggcgccgggggaggagcggcggcgcagcccgctggaccacctgcctccgcctgccaactccaacaagccactgacgccgttcagcatcgaggacatcctcaacaagccgtctgtgcggagaagttactcgctgtgcggggcggcgcacctgctggccgccgcggacaagcacgcgcagggcggcttgcccctggcgggccgcgcgctgctctcgcagacctcgccgctgtgcgcgctggaggagctcgccagcaagacgtttaaggggctggaggtcagcgttctgcaggcagccgaaggccgcgacggtatgaccatctttgggcagcggcagacccctaagaagcggcgaaagtcgcgcacggccttcaccaaccaccagatctatgaattggaaaagcgctttctataccagaagtacctgtcccccgccgatcgcgaccaaatcgcgcagcagctgggcctcaccaacgcgcaagtcatcacctggttccagaatcggcgcgctaagctcaagcgggacctggaggagatgaaggccgacgtagagtccgccaagaaactgggccccagcgggcagatggacatcgtggcgctggccgaactcgagcagaactcggaggccacagccggcggtggcggcggctgcggcagggccaagtcgaggcccggctctccggtcctccccccaggcgccccgaaggccccgggcgctggcgccctgcagctctcgcctgcctctccgctcacggaccagccggccagcagccaggactgctcggaggacgaggaagacgaagagatcgacgtggacgattga; NM_010691.(SEQ ID NO: 59) Lmx1b:atgttggacggcatcaagatggaggagcacgccctgcgccccgggcccgccactctgggggtgctgctgggctccgactgcccgcatcccgccgtctgcgagggctgccagcggcccatctccgaccgcttcctgatgcgagtcaacgagtcgtcctggcacgaggagtgtttgcagtgcgcggcgtgtcagcaagccctcaccaccagctgctacttccgggatcggaaactgtactgcaaacaagactaccaacagctcttcgcggccaagtgcagcggctgcatggagaagatcgcccccaccgagttcgtgatgcgggcgctggagtgcgtgtaccacctgggctgcttctgctgctgcgtgtgtgaacggcagctacgcaagggcgacgaattcgtgctcaaggagggccagctgctgtgcaagggtgactacgagaaggagaaggacctgctcagctccgtgagccccgacgagtccgactccgtgaagagcgaggatgaagatggggacatgaagccggccaaggggcagggcagtcagagcaagggcagcggggatgacgggaaggacccgcggaggcccaagcgaccccggaccatcctcaccacgcagcagcgaagagccttcaaggcctccttcgaggtctcgtcgaagccttgccgaaaggtccgagagacactggcagctgagacgggcctcagtgtgcgcgtggtccaggtctggtttcagaaccaaagagcaaagatgaagaagctggcgcggcggcaccagcagcagcaggagcagcagaactcccagcggctgggccaggaggtcctgtccagccgcatggagggcatgatggcttcctacacgccgctggccccaccacagcagcagatcgtggccatggaacagagcccctacggcagcagcgaccccttccagcagggcctcacgccgccccaaatgccagggaacgactccatcttccatgacatcgacagcgatacctccttaaccagcctcagcgactgcttcctcggctcctcagacgtgggctccctgcaggcccgcgtggggaaccccatcgaccggctctactccatgcagagttcctacttcgcctcctga; NM 010725. (SEQ ID NO: 60) Neurogenin(NEUROG1): NM_006161:atgccagcccgccttgagacctgcatctccgacctcgactgcgccagcagcagcggcagtgacctatccggcttcctcaccgacgaggaagactgtgccagactccaacaggcagcctccgcttcggggccgcccgcgccggcccgcaggggcgcgcccaatatctcccgggcgtctgaggttccaggggcacaggacgacgagcaggagaggcggcggcgccgcggccggacgcgggtccgctccgaggcgctgctgcactcgctgcgcaggagccggcgcgtcaaggccaacgatcgcgagcgcaaccgcatgcacaacttgaacgcggccctggacgcactgcgcagcgtgctgccctcgttccccgacgacaccaagctcaccaaaatcgagacgctgcgcttcgcctacaactacatctgggctctggccgagacactgcgcctggcggatcaagggctgcccggaggcggtgcccgggagcgcctcctgccgccgcagtgcgtcccctgcctgcccggtcccccaagccccgccagcgacgcggagtcctggggctcaggtgccgccgccgcctccccgctctctgaccccagtagcccagccgcctccgaagacttcacctaccgccccggcgaccctgttttctccttcccaagcctgcccaaagacttgctccacacaacgccctgtttcattccttaccactag;BQ169355. (SEQ ID NO: 61)Neurogenin2(NEUROG2): NM024019: atgttcgtcaaatccgagaccttggagttgaaggaggaagaggacgtgttagtgctgctcggatcggcctcccccgccttggcggccctgaccccgctgtcatccagcgccgacgaagaagaggaggaggagccgggcgcgtcaggcggggcgcgtcggcagcgcggggctgaggccgggcagggggcgcggggcggcgtggctgcgggtgcggagggctgccggcccgcacggctgctgggtctggtacacgattgcaaacggcgcccttcccgggcgcgggccgtctcccgaggcgccaagacggccgagacggtgcagcgcatcaagaagacccgtagactgaaggccaacaaccgcgagcgaaaccgcatgcacaacctcaacgcggcactggacgcgctgcgcgaggtgctccccacgttccccgaggacgccaagctcaccaagatcgagaccctgcgcttcgcccacaactacatctgggcactcaccgagaccctgcgcctggcggatcactgcgggggcggcggcgggggcctgccgggggcgctcttctccgaggcagtgtlgctgagcccgggaggcgccagcgccgccctgagcagcagcggagacagcccctcgcccgcctccacgtggagttgcaccaacagccccgcgccgtcctcctccgtglcctccaattccacctccccctacagctgcactttatcgcccgccagcccggccgggtcagacatggactattggcagcccccacctcccgacaagcaccgctatgcacctcacctccccatagccagggattgtatctag; DR001447.(SEQ ID NO: 62)Neurogenin3(NEUROG3): atgacgcctcaaccctcgggtgcgcccactgtccaagtgacccgtgagacggagcggtccttccccagagcctcggaagacgaagtgacctgccccacgtccgccccgcccagccccactcgcacacgggggaactgcgcagaggcggaagagggaggctgccgaggggccccgaggaagctccgggcacggcgcgggggacgcagccggcctaagagcgagttggcactgagcaagcagcgacggagtcggcgaaagaaggccaacgaccgcgagcgcaatcgaatgcacaacctcaactcggcactggacgccctgcgcggtgtcctgcccaccttcccagacgacgcgaagctcaccaagatcgagacgctgcgcttcgcccacaactacatctgggcgctgactcaaacgctgcgcatagcggaccacagcttgtacgcgctggagccgccggcgccgcactgcggggagctgggcagcccaggcggttcccccggggactgggggtccctctactccccagtctcccaggctggcagcctgagtcccgccgcgtcgctggaggagcgacccgggctgctgggggccaccttttccgcctgcttgagcccaggcagtctggctttctcagattttctgtga; NM 009719. (SEQ ID NO: 63)MASH1: NM_004316:atggaaagctctgccaagatggagagcggcggcgccggccagcagccccagccgcagccccagcagcccttcctgccgcccgcagcctgtttctttgccacggccgcagccgcggcggccgcagccgccgcagcggcagcgcagagcgcgcagcagcagcagcagcagcagcagcagcagcagcaggcgccgcagctgagaccggcggccgacggccagccctcagggggcggtcacaagtcagcgcccaagcaagtcaagcgacagcgctcgtcttcgcccgaactgatgcgctgcaaacgccggctcaacttcagcggctttggctacagcctgccgcagcagcagccggccgccgtggcgcgccgcaacgagcgcgagcgcaaccgcgtcaagttggtcaacctgggctttgccacccttcgggagcacgtccccaacggcgcggccaacaagaagatgagtaaggtggagacactgcgctcggcggtcgagtacatccgcgcgctgcagcagctgctggacgagcatgacgcggtgagcgccgccttccaggcaggcgtcctgtcgcccaccatctcccccaactactccaacgacttgaactccatggccggctcgccggtctcatcctactcgtcggacgagggctcttacgacccgctcagccccgaggagcaggagcttctcgacttcaccaactggttctga; NM 008553. (SEQ ID NO: 64)MyoD: NM010866: atggagcttctatcgccgccactccgggacatagacttgacaggccccgacggctctctctgctcctttgagacagcagacgacttctatgatgacccgtgtttcgactcaccagacctgcgcttttttgaggacctggacccgcgcctggtgcacatgggagccctcctgaaaccggaggagcacgcacacttccctactgcggtgcacccaggcccaggcgctcgtgaggatgagcatgtgcgcgcgcccagcgggcaccaccaggcgggtcgctgcttgctgtgggcctgcaaggcgtgcaagcgcaagaccaccaacgctgatcgccgcaaggccgccaccatgcgcgagcgccgccgcctgagcaaagtgaatgaggccttcgagacgctcaagcgctgcacgtccagcaacccgaaccagcggctacccaaggtggagatcctgcgcaacgccatccgctacatcgaaggtctgcaggctctgctgcgcgaccaggacgccgcgccccctggcgccgctgccttctacgcacctggaccgctgcccccaggccgtggcagcgagcactacagtggcgactcagatgcatccagcccgcgctccaactgctctgatggcatgatggattacagcggccccccaagcggcccccggcggcagaatggctacgacaccgcctactacagtgaggcggcgcgcgagtccaggccagggaagagtgcggctgtgtcgagcctcgactgcctgtccagcatagtggagcgcatctccacagacagccccgctgcgcctgcgctgcttttggcagatgcaccaccagagtcgcctccgggtccgccagagggggcatccctaagcgacacagaacagggaacccagaccccgtctcccgacgccgcccctcagtgtcctgcaggctcaaaccccaatgcgatttatcaggtgctttga; (SEQ ID NO: 65)NM002478: atggagctactgtcgccaccgctccgcgacgtagacctgacggcccccgacggctctctctgctcctttgccacaacggacgacttctatgacgacccgtgtttcgactccccggacctgcgcttcttcgaagacctggacccgcgcctgatgcacgtgggcgcgctcctgaaacccgaagagcactcgcacttccccgcggcggtgcacccggccccgggcgcacgtgaggacgagcatgtgcgcgcgcccagcgggcaccaccaggcgggccgctgcctactgtgggcctgcaaggcgtgcaagcgcaagaccaccaacgccgaccgccgcaaggccgccaccatgcgcgagcggcgccgcctgagcaaagtaaatgaggcctttgagacactcaagcgctgcacgtcgagcaatccaaaccagcggttgcccaaggtggagatcctgcgcaacgccatccgctatatcgagggcctgcaggctctgctgcgcgaccaggacgccgcgccccctggcgccgcagccgccttctatgcgccgggcccgctgcccccgggccgcggcggcgagcactacagcggcgactccgacgcgtccagcccgcgctccaactgctccgacggcatgatggactacagcggccccccgagcggcgcccggcggcggaactgctacgaaggcgcctaclacaacgagactgcccgccctcctgctggcggacgtgccttctgagtcgcctccgcgcaggcaagaggctgccgcccccagcgagggagagagcagcggcgaccccacccagtcaccggacgccgccccgcagtgccctgcgggtgcgaaccccaacccgatataccaggtgctctga. (SEQ ID NO: 66)Myf5: NM_005593: atggacgtgatggatggctgccagttctcaccttctgagtacttctacgacggctcctgcataccgtcccccgagggtgaatttggggacgagtttgtgccgcgagtggctgccttcggagcgcacaaagcagagctgcagggctcagatgaggacgagcacgtgcgagcgcctaccggccaccaccaggctggtcactgcctcatgtgggcctgcaaagcctgcaagaggaagtccaccaccatggatcggcggaaggcagccactatgcgcgagcggaggcgcctgaagaaggtcaaccaggctttcgaaaccctcaagaggtgtaccacgaccaaccccaaccagaggctgcccaaggtggagatcctcaggaatgccatccgctacatcgagagcctgcaggagttgctgagagagcaggtggagaactactatagcctgccgggacagagctgctcggagcccaccagccccacctccaactgctctgatggcatgcccgaatgtaacagtcctgtctggtccagaaagagcagtacttttgacagcatctactgtcctgatgtatcaaatgtatatgccacagataaaaactccttatccagcttggattgcttatccaacatagtggaccggatcacctcctcagagcaacctgggttgcctctccaggatctggcttctctctctccagttgccagcaccgattcacagcctgcaactccaggggcttctagttccaggcttatctatcatgtgctatga; NM  131576; NM_008656.(SEQ ID NO: 67)Myf6: NM_002469: atgatgatggacctttttgaaactggctcctatttcttctacttggatggggaaaatgttactctgcagccattagaagtggcagaaggctctcctttgtatccagggagtgatggtaccttgtccccctgccaggaccaaatgcccccggaagcggggagcgacagcagcggagaggaacatgtcctggcgcccccgggcctgcagcctccacactgccccggccagtgtctgatctgggcttgcaagacctgcaagagaaaatctgcccccactgaccggcgaaaagccgccaccctgcgcgaaaggaggaggctaaagaaaatcaacgaggccttcgaggcactgaagcggcgaactgtggccaaccccaaccagaggctgcccaaggtggagattctgcggagcgccatcagctatattgagcggctgcaggacctgctgcaccggctggatcagcaggagaagatgcaggagctgggggtggaccccttcagctacagacccaaacaagaaaatcttgagggtgcggatttcctgcgcacctgcagctcccagtggccaagtgtttccgatcattccagggggctcgtgataacggctaaggaaggaggagcaagtattgattcgtcagcctcgagtagccttcgatgcctttcttccatcgtggacagtatttcctcggaggaacgcaaactcccctgcgtggaggaagtggtggagaagtaa; NM_008657; NM_008657; NMJ) 13172. (SEQ ID NO: 68)Ifrd1: NM_001007245:atgccgaagaacaagaagcggaacactccccaccgcggtagcagtgctggcggcggcgggtcaggagcagccgcagcgacggcggcgacagcaggtggccagcatcgaaatgttcagccttttagtgatgaagatgcatcaattgaaacaatgagccattgcagtggttatagcgatccttccagttttgctgaagatggaccagaagtccttgatgaggaaggaactcaagaagacctagagtacaagttgaagggattaattgacctaaccctggataagagtgcgaagacaaggcaagcagctcttgaaggtattaaaaatgcactggcttcaaaaatgctgtatgaatttattctggaaaggagaatgactttaactgatagcattgaacgctgcctgaaaaaaggtaagagtgatgagcaacgtgcagctgcagcgttagcatctgttctttgtattcagctgggccctggaattgaaagtgaagagattttgaaaactcttggaccaatcctaaagaaaatcatttgtgatgggtcagctagtatgcaggctaggcaaacttgtgcaacttgctttggtgtttgctgttttattgccacagatgacattactgaactatactcaactctggaatgtttggaaaatatcttcactaaatcctatctcaaagagaaagacactactgttatttgcagcactcctaatacagtgcttcatatcagctctcttcttgcatggacactactgctgaccatatgcccaatcaatgaagtgaagaaaaagcttgagatgcatttccataagcttccaagcctcctctcttgtgatgatgtaaacatgagaatagctgctggtgaatctttggcacttctctttgaattggccagaggaatagagagtgactttttttatgaagacatggagtccttgacgcagatgcttagggccttggcaacagatggaaataaacaccgggccaaagtggacaagagaaagcagcggtcagttttcagagatgtcctgagggcagtggaggaacgggattttccaacagaaaccattaaatttggtcctgaacgcatgtatattgattgctgggtaaaaaaacacacctatgacacctttaaggaggttcttggatcagggatgcagtaccacttgcagtcaaatgaattccttcgaaatgtatttgaacttggacccccagtgatgcttgatgctgcaacgcttaaaacgatgaagatttctcgtttcgaaaggcatttatataactctgcagccttcaaagctcgaaccaaagctagaagcaaatgtcgagataagagagcagatgttggagaattcttctag. (SEQ ID NO: 69)MeGA: NM_013172: atggggcggaagaaaatacaaatcacacgcataatggatgaaaggaaccgacaggtcacttttacaaagagaaagtttggattaatgaagaaagcctatgaacttagtgtgctctgtgactgtgaaatagcactcatcattttcaacagctctaacaaactgtttcaatatgctagcactgatatggacaaagttcttctcaagtatacagaatataatgaacctcatgaaagcagaaccaactcggatattgttgaggctctgaacaagaaggaacacagagggtgcgacagcccagaccctgatacttcatatgtgctaactccacatacagaagaaaaatataaaaaaattaatgaggaatttgataatatgatgcggaatcataaaatcgcacctggtctgccacctcagaacttttcaatgtctgtcacagttccagtgaccagccccaatgctttgtcctacactaacccagggagttcactggtgtccccatctttggcagccagctcaacgttaacagattcaagcatgctctctccacctcaaaccacattacatagaaatgtgtctcctggagctcctcagagaccaccaagtactggcaatgcaggtgggatgttgagcactacagacctcacagtgccaaatggagctggaagcagtccagtggggaatggatttgtaaactcaagagcttctccaaatttgattggagctactggtgcaaatagcttaggcaaagtcatgcctacaaagtctccccctccaccaggtggtggtaatcttggaatgaacagtaggaaaccagatcttcgagttgtcatccccccttcaagcaagggcatgatgcctccactatcggaggaagaggaattggagttgaacacccaaaggatcagtagttctcaagccactcaacctcttgctaccccagtcgtgtctgtgacaaccccaagcttgcctccgcaaggacttgtgtactcagcaatgccgactgcctacaacactgattattcactgaccagcgctgacctgtcagcccttcaaggcttcaactcgccaggaatgctgtcgctgggacaggtgtcggcctggcagcagcaccacctaggacaagcagccctcagctctcttgttgctggagggcagttatctcagggttccaatttatccattaataccaaccaaaacatcagcatcaagtccgaaccgatttcacctcctcgggatcgtatgaccccatcgggcttccagcagcagcagcagcagcagcagcagcagcagccgccgccaccaccgcagccccagccacaacccccgcagccccagccccgacaggaaatggggcgctcccctgtggacagtctgagcagctctagtagctcctatgatggcagtgatcgggaggatccacggggcgacttccattctccaattgtgcttggccgacccccaaacactgaggacagagaaagcccttctgtaaagcgaatgaggatggacgcgtgggtgacctaa.(SEQ ID NO: 70)Myogenin: NM_002479: atggagctgtatgagacatccccctacttctaccaggaaccccgcttctatgatggggaaaactacctgcctgtccacctccagggcttcgaaccaccaggctacgagcggacggagctcaccctgagccccgaggccccagggccccttgaggacaaggggctggggacccccgagcactgtccaggccagtgcctgccgtgggcgtgtaaggtgtgtaagaggaagtcggtgtccgtggaccggcggcgggcggccacactgagggagaagcgcaggctcaagaaggtgaatgaggccttcgaggccctgaagagaagcaccctgctcaaccccaaccagcggctgcccaaggtggagatcctgcgcagtgccatccagtacatcgagcgcctccaggccctgctcagctccctcaaccaggaggagcgtgacctccgctaccggggcgggggcgggccccagccaggggtgcccagcgaatgcagctctcacagcgcctcctgcagtccagagtggggcagtgcactggagttcagcgccaacccaggggatcatctgctcacggctgaccctacagatgcccacaacctgcactccctcacctccatcgtggacagcatcacagtggaagatgtgtctgtggccttcccagatgaaaccatgcccaactga;BC053899; BC068019; AB257560. (SEQ ID NO: 71)Nkx2.2: NM_002509: atgtcgctgaccaacacaaagacggggttttcggtcaaggacatcttagacctgccggacaccaacgatgaggagggctctgtggccgaaggtccggaggaagagaacgaggggcccgagccagccaagagggccgggccgctggggcagggcgccctggacgcggtgcagagcctgcccctgaagaaccccttctacgacagcagcgacaacccgtacacgcgctggctggccagcaccgagggccttcagtactccctgcacggtctggctgccggggcgccccctcaggactcaagctccaagtccccggagccctcggccgacgagtcaccggacaatgacaaggagaccccgggcggcgggggggacgccggcaagaagcgaaagcggcgagtgcttttctccaaggcgcagacctacgagctggagcggcgctttcggcagcagcggtacctgtcggcgcccgagcgcgaacacctggccagcctcatccgcctcacgcccacgcaggtcaagatctggttccagaaccaccgctacaagatgaagcgcgcccgggccgagaaaggtatggaggtgacgcccctgccctcgccgcgccgggtggccgtgcccgtcttggtcagggacggcaaaccatgtcacgcgctcaaagcccaggacctggcagccgccaccttccaggcgggcattcccttttctgcctacagcgcgcagtcgctgcagcacatgcagtacaacgcccagtacagctcggccagcaccccccagtacccgacagcacaccccctggtccaggcccagcagtggacttggtga; NM  001077632; NMJ) 10919.Notch (SEQ ID NO: 72) Notch1: NM_017617:atgccgccgctcctggcgcccctgctctgcctggcgctgctgcccgcgctcgccgcacgaggcccgcgatgctcccagcccggtgagacctgcctgaatggcgggaagtgtgaagcggccaatggcacggaggcctgcgtctgtggcggggccttcgtgggcccgcgatgccaggaccccaacccgtgcctcagcaccccctgcaagaacgccgggacatgccacgtggtggaccgcagaggcgtggcagactatgcctgcagctgtgccctgggcttctctgggcccctctgcctgacacccctggacaatgcctgcctcaccaacccctgccgcaacgggggcacctgcgacctgctcacgctgacggagtacaagtgccgctgcccgcccggctggtcagggaaatcgtgccagcaggctgacccgtgcgcctccaacccctgcgccaacggtggccagtgcctgcccttcgaggcctcctacatctgccactgcccacccagcttccatggccccacctgccggcaggatgtcaacgagtgtggccagaagcccgggctttgccgccacggaggcacctgccacaacgaggtcggctcctaccgctgcgtctgccgcgccacccacactggccccaactgcgagcggccctacgtgccctgcagcccctcgccctgccagaacgggggcacctgccgccccacgggcgacgtcacccacgagtgtgcctgcctgccaggcttcaccggccagaactgtgaggaaaatatcgacgattgtccaggaaacaactgcaagaacgggggtgcctgtgtggacggcgtgaacacctacaactgccgctgcccgccagagtggacaggtcagtactgtaccgaggatgtggacgagtgccagctgatgccaaatgcctgccagaacggcgggacctgccacaacacccacggtggctacaactgcgtgtgtgtcaacggctggactggtgaggactgcagcgagaacattgatgactgtgccagcgccgcctgcltccacggcgccacctgccatgaccgtgtggcctccttctactgcgagtgtccccatggccgcacaggtctgctgtgccacclcaacgacgcatgcatcagcaacccctgtaacgagggctccaactgcgacaccaaccctgtcaatggcaaggccatctgcacctgcccctcggggtacacgggcccggcctgcagccaggacgtggatgagtgctcgctgggtgccaacccctgcgagcatgcgggcaagtgcatcaacacgctgggctccttcgagtgccagtgtctgcagggctacacgggcccccgatgcgagatcgacgtcaacgagtgcgtctcgaacccgtgccagaacgacgccacctgcctggaccagattggggagttccagtgcatctgcatgcccggctacgagggtgtgcactgcgaggtcaacacagacgagtgtgccagcagcccctgcctgcacaatggccgctgcctggacaagatcaatgagttccagtgcgagtgccccacgggcttcactgggcatctgtgccagtacgatgtggacgagtgtgccagcaccccctgcaagaatggtgccaagtgcctggacggacccaacacttacacctgtgtgtgcacggaagggtacacggggacgcactgcgaggtggacatcgatgagtgcgaccccgacccctgccactacggctcctgcaaggacggcgtcgccaccttcacctgcctctgccgcccaggctacacgggccaccaclgcgagaccaacatcaacgagtgctccagccagccctgccgccacgggggcacctgccaggaccgcgacaacgcctacctctgcltctgcctgaaggggaccacaggacccaactgcgagatcaacctggatgactgtgccagcagcccctgcgactcgggcacctgtctggacaagatcgatggctacgagtgtgcctgtgagccgggctacacagggagcatgtgtaacatcaacatcgatgagtgtgcgggcaacccctgccacaacgggggcacctgcgaggacggcatcaatggcttcacctgccgctgccccgagggctaccacgaccccacctgcctgtctgagglcaatgagtgcaacagcaacccctgcgtccacggggcctgccgggacagcctcaacgggtacaagtgcgactgtgaccctgggtggagtgggaccaactgtgacatcaacaacaatgagtgtgaatccaacccttgtgtcaacggcggcacctgcaaagacatgaccagtggctacgtgtgcacctgccgggagggcttcagcggtcccaactgccagaccaacatcaacgagtgtgcgtccaacccatgtctgaaccagggcacgtgtattgacgacgttgccgggtacaagtgcaactgcctgctgccctacacaggtgccacgtgtgaggtgglgctggccccgtgtgcccccagcccctgcagaaacggcggggagtgcaggcaatccgaggactatgagagcttctcctgtgtctgccccacgggctggcaagggcagacctgtgagglcgacatcaacgagtgcgttctgagcccgtgccggcacggcgcatcctgccagaacacccacggcggctaccgclgccactgccaggccggctacagtgggcgcaactgcgagaccgacatcgacgactgccggcccaacccgtgtcacaacgggggctcctgcacagacggcatcaacacggccttclgcgactgcctgcccggcttccggggcactttctgtgaggaggacatcaacgagtgtgccagtgacccctgccgcaacggggccaactgcacggactgcgtggacagctacacgtgcacctgccccgcaggcttcagcgggatccactgtgagaacaacacgcctgactgcacagagagctcctgcttcaacggtggcacctgcgtggacggcatcaactcgttcacctgcctgtgtccacccggcttcacgggcagctactgccagcacgatgtcaatgagtgcgactcacagccctgcctgcatggcggcacctgtcaggacggctgcggctcctacaggtgcacctgcccccagggctacactggccccaactgccagaaccttgtgcactggtgtgactcctcgccctgcaagaacggcggcaaatgctggcagacccacacccagtaccgctgcgagtgccccagcggctggaccggcctttactgcgacgtgcccagcgtgtcctgtgaggtggctgcgcagcgacaaggtgttgacgttgcccgcctgtgccagcatggagggctctgtgtggacgcgggcaacacgcaccactgccgctgccaggcgggctacacaggcagctactgtgaggacctggtggacgagtgctcacccagcccctgccagaacggggccacctgcacggactacctgggcggctactcctgcaagtgcgtggccggctaccacggggtgaactgctctgaggagatcgacgagtgcctctcccacccctgccagaacgggggcacctgcctcgacctccccaacacctacaagtgctcctgcccacggggcactcagggtgtgcactgtgagatcaacgtggacgactgcaatccccccgttgaccccgtgtcccggagccccaagtgctttaacaacggcacctgcgtggaccaggtgggcggctacagctgcacctgcccgccgggcttcgtgggtgagcgctgtgagggggatgtcaacgagtgcctgtccaatccctgcgacgcccgtggcacccagaactgcgtgcagcgcgtcaatgacttccactgcgagtgccgtgctggtcacaccgggcgccgctgcgagtccgtcatcaatggctgcaaaggcaagccctgcaagaatgggggcacctgcgccgtggcctccaacaccgcccgcgggttcatctgcaagtgccctgcgggcttcgagggcgccacgtgtgagaatgacgctcgtacctgcggcagcctgcgctgcctcaacggcggcacatgcatctccggcccgcgcagccccacctgcctgtgcctgggccccttcacgggccccgaatgccagttcccggccagcagcccctgcctgggcggcaacccctgctacaaccaggggacctgtgagcccacatccgagagccccttctaccgttgcctgtgccccgccaaattcaacgggctcttgtgccacatcctggactacagcttcgggggtggggccgggcgcgacatccccccgccgctgatcgaggaggcgtgcgagctgcccgagtgccaggaggacgcgggcaacaaggtclgcagcctgcagtgcaacaaccacgcgtgcggctgggacggcggtgactgctccctcaacttcaatgacccctggaagaactgcacgcagtctctgcagtgctggaagtacttcagtgacggccactgtgacagccagtgcaactcagccggctgcctcttcgacggctttgactgccagcgtgcggaaggccagtgcaaccccctgtacgaccagtactgcaaggaccacttcagcgacgggcactgcgaccagggctgcaacagcgcggaglgcgagtgggacgggctggactgtgcggagcatgtacccgagaggctggcggccggcacgctggtggtggtggtgctgatgccgccggagcagctgcgcaacagclccttccacttcctgcgggagctcagccgcgtgctgcacaccaacgtggtcttcaagcgtgacgcacacggccagcagatgatcttcccctactacggccgcgaggaggagctgcgcaagcaccccatcaagcgtgccgccgagggctgggccgcacctgacgccctgctgggccaggtgaaggcctcgctgctccctggtggcagcgagggtgggcggcggcggagggagctggaccccatggacgtccgcggctccatcgtctacctggagattgacaaccggcagtgtgtgcaggcctcctcgcagtgcttccagagtgccaccgacgtggccgcattcctgggagcgctcgcctcgctgggcagcctcaacatcccctacaagatcgaggccgtgcagagtgagaccgtggagccgcccccgccggcgcagctgcacttcatgtacgtggcggcggccgcctttgtgcttctgttcttcgtgggctgcggggtgctgctgtcccgcaagcgccggcggcagcatggccagctctggttccctgagggcttcaaagtgtctgaggccagcaagaagaagcggcgggagcccctcggcgaggactccgtgggcctcaagcccctgaagaacgcttcagacggtgccctcatggacgacaaccagaatgagtggggggacgaggacctggagaccaagaagttccggttcgaggagcccgtggttctgcctgacctggacgaccagacagaccaccggcagtggactcagcagcacctggatgccgctgacctgcgcatgtctgccatggcccccacaccgccccagggtgaggttgacgccgactgcatggacgtcaatgtccgcgggcctgatggcttcaccccgctcatgatcgcctcctgcagcgggggcggcctggagacgggcaacagcgaggaagaggaggacgcgccggccgtcatctccgacttcatctaccagggcgccagcctgcacaaccagacagaccgcacgggcgagaccgccttgcacctggccgcccgctactcacgctctgatgccgccaagcgcctgctggaggccagcgcagatgccaacatccaggacaacatgggccgcaccccgctgcatgcggctgtgtctgccgacgcacaaggtgtcttccagatcctgatccggaaccgagccacagacctggatgcccgcatgcatgatggcacgacgccactgatcctggctgcccgcctggccgtggagggcatgctggaggacctcatcaactcacacgccgacgtcaacgccgtagatgacctgggcaagtccgccctgcactgggccgccgccgtgaacaatgtggatgccgcagttgtgctcctgaagaacggggctaacaaagatatgcagaacaacagggaggagacacccctgtttctggccgcccgggagggcagctacgagaccgccaaggtgctgctggaccactttgccaaccgggacatcacggatcatatggaccgcctgccgcgcgacatcgcacaggagcgcatgcatcacgacatcgtgaggctgctggacgagtacaacctggtgcgcagcccgcagctgcacggagccccgctggggggcacgcccaccctgtcgcccccgctctgctcgcccaacggctacctgggcagcctcaagcccggcgtgcagggcaagaaggtccgcaagcccagcagcaaaggcctggcctgtggaagcaaggaggccaaggacctcaaggcacggaggaagaagtcccaggacggcaagggctgcctgctggacagctccggcatgctctcgcccgtggactccctggagtcaccccatggctacctgtcagacgtggcctcgccgccactgctgccctccccgttccagcagtctccgtccgtgcccctcaaccacctgcctgggatgcccgacacccacctgggcatcgggcacctgaacgtggcggccaagcccgagatggcggcgctgggtgggggcggccggctggcctttgagactggcccacctcgtctctcccacctgcctgtggcctctggcaccagcaccgtcctgggctccagcagcggaggggccctgaatttcactgtgggcgggtccaccagtttgaatggtcaatgcgagtggctgtcccggctgcagagcggcatggtgccgaaccaatacaaccctctgcgggggagtgtggcaccaggccccctgagcacacaggccccctccctgcagcatggcatggtaggcccgctgcacagtagccttgctgccagcgccctgtcccagatgatgagctaccagggcctgcccagcacccggctggccacccagcctcacctggtgcagacccagcaggtgcagccacaaaacttacagatgcagcagcagaacctgcagccagcaaacatccagcagcagcaaagcctgcagccgccaccaccaccaccacagccgcaccttggcgtgagctcagcagccagcggccacctgggccggagcttcctgagtggagagccgagccaggcagacgtgcagccactgggccccagcagcctggcggtgcacactattctgccccaggagagccccgccctgcccacgtcgctgccatcctcgctggtcccacccgtgaccgcagcccagttcctgacgcccccctcgcagcacagctactcctcgcctgtggacaacacccccagccaccagctacaggtgcctgagcaccccttcctcaccccgtcccctgagtcccctgaccagtggtccagctcgtccccgcattccaacgtctccgactggtccgagggcgtctccagccctcccaccagcatgcagtcccagatcgcccgcattccggaggccttcaagtaa; AFI 59231.NOTCH2: NM 024408; NMJ) 10928. (SEQ ID NO: 73)NOTCH3: NM000435: atggggccgggggcccgtggccgccgccgccgccgtcgcccgatgtcgccgccaccgccaccgccacccgtgcgggcgctgcccctgctgctgctgctagcggggccgggggctgcagcccccccttgcctggacggaagcccgtgtgcaaatggaggtcgttgcacccagctgccctcccgggaggctgcctgcctgtgcccgcctggctgggtgggtgagcggtgtcagctggaggacccctgtcactcaggcccctgtgctggccgtggtgtctgccagagttcagtggtggctggcaccgcccgattctcatgccggtgcccccgtggcttccgaggccctgactgctccctgccagatccctgcctcagcagcccttgtgcccacggtgcccgctgctcagtggggcccgatggacgcttcctctgctcctgcccacctggctaccagggccgcagctgccgaagcgacgtggatgagtgccgggtgggtgagccctgccgccatggtggcacctgcctcaacacacctggctccttccgctgccagtgtccagctggctacacagggccactatgtgagaaccccgcggtgccctgtgcaccctcaccatgccgtaacgggggcacctgcaggcagagtggcgacctcacttacgactgtgcctgtcttcctgggtttgagggtcagaattgtgaagtgaacgtggacgactgtccaggacaccgatgtctcaatggggggacatgcgtggatggcgtcaacacctataactgccagtgccctcctgagtggacaggccagttctgcacggaggacgtggatgagtgtcagctgcagcccaacgcctgccacaatgggggtacctgcttcaacacgctgggtggccacagctgcgtgtgtgtcaatggctggacaggcgagagctgcagtcagaatatcgatgactgtgccacagccgtgtgcttccatggggccacctgccatgaccgcgtggcttctttctactgtgcctgccccatgggcaagactggcctcctgtgtcacctggatgacgcctgtgtcagcaacccctgccacgaggatgctatctgtgacacaaatccggtgaacggccgggccatttgcacctgtcctcccggcttcacgggtggggcatgtgaccaggatgtggacgagtgctctatcggcgccaacccctgcgagcacttgggcaggtgcgtgaacacgcagggctccttcctgtgccagtgcggtcgtggctacactggacctcgctgtgagaccgatgtcaacgagtgtctgtcggggccctgccgaaaccaggccacgtgcctcgaccgcataggccagttcacctgtatctgtatggcaggcttcacaggaacctattgcgaggtggacattgacgagtgtcagagtagcccctgtgtcaacggtggggtctgcaaggaccgagtcaatggcttcagctgcacctgcccctcgggcttcagcggctccacgtgtcagctggacgtggacgaatgcgccagcacgccctgcaggaatggcgccaaatgcgtggaccagcccgatggctacgagtgccgctgtgccgagggctttgagggcacgctgtgtgatcgcaacgtggacgactgctcccctgacccatgccaccatggtcgctgcgtggatggcatcgccagcttctcatgtgcctgtgctcctggctacacgggcacacgctgcgagagccaggtggacgaatgccgcagccagccctgccgccatggcggcaaatgcctagacctggtggacaagtacctctgccgctgcccttctgggaccacaggtgtgaactgcgaagtgaacattgacgactgtgccagcaacccctgcacctttggagtctgccgtgatggcatcaaccgctacgactgtgtctgccaacctggcttcacagggcccctttgtaacgtggagatcaatgagtgtgcttccagcccatgcggcgagggaggttcctgtgtggatggggaaaatggcttccgctgcctctgcccgcctggctccttgcccccactctgcctccccccgagccatccctgtgcccatgagccctgcagtcacggcatctgctatgatgcacctggcgggttccgctgtgtgtgtgagcctggctggagtggcccccgctgcagccagagcctggcccgagacgcctgtgagtcccagccgtgcagggccggtgggacatgcagcagcgatggaatgggtttccactgcacctgcccgcctggtgtccagggacgtcagtgtgaactcctctccccctgcaccccgaacccctgtgagcatgggggccgctgcgagtctgcccctggccagctgcctgtctgctcctgcccccagggctggcaaggcccacgatgccagcaggatgtggacgagtgtgctggccccgcaccctgtggccctcatggtatctgcaccaacctggcagggagtttcagctgcacctgccatggagggtacactggcccttcctgcgatcaggacatcaatgactgtgaccccaacccatgcctgaacggtggctcgtgccaagacggcgtgggctccttttcctgctcctgcctccctggtttcgccggcccacgatgcgcccgcgatgtggatgagtgcctgagcaacccctgcggcccgggcacctgtaccgaccacgtggcctccttcacctgcacctgcccgccaggctacggaggcttccactgcgaacaggacctgcccgactgcagccccagctcctgcttcaatggcgggacctgtgtggacggcgtgaactcgttcagctgcctgtgccgtcccggctacacaggagcccactgccaacatgaggcagacccctgcctctcgcggccctgcctacacgggggcgtctgcagcgccgcccaccctggcttccgctgcacctgcctcgagagcttcacgggcccgcagtgccagacgctggtggattggtgcagccgccagccttgtcaaaacgggggtcgctgcgtccagactggggcctattgcctttgtccccctggatggagcggacgcctctgtgacatccgaagcttgccctgcagggaggccgcagcccagatcggggtgcggctggagcagctgtgtcaggcgggtgggcagtgtgtggatgaagacagctcccactactgcgtgtgcccagagggccgtactggtagccactgtgagcaggaggtggacccctgcttggcccagccctgccagcatggggggacctgccgtggctatatggggggctacatgtgtgagtgtcttcctggctacaatggtgataactgtgaggacgacgtggacgagtgtgcctcccagccctgccagcacgggggttcatgcattgacctcgtggcccgctatctctgctcctgtcccccaggaacgctgggggtgctctgcgagattaatgaggatgactgcggcccaggcccaccgctggactcagggccccggtgcctacacaatggcacctgcgtggacctggtgggtggtttccgctgcacctgtcccccaggatacactggtttgcgctgcgaggcagacatcaatgagtgtcgctcaggtgcctgccacgcggcacacacccgggactgcctgcaggacccaggcggaggtttccgttgcctttgtcatgctggcttctcaggtcctcgctgtcagactgtcctgtctccctgcgagtcccagccatgccagcatggaggccagtgccgtcctagcccgggtcctgggggtgggctgaccttcacctgtcactgtgcccagccgttctggggtccgcgttgcgagcgggtggcgcgctcctgccgggagctgcagtgcccggtgggcgtcccatgccagcagacgccccgcgggccgcgctgcgcctgccccccagggttgtcgggaccctcctgccgcagcttcccggggtcgccgccgggggccagcaacgccagctgcgcggccgccccctgtctccacgggggctcctgccgccccgcgccgctcgcgcccttcttccgctgcgcttgcgcgcagggctggaccgggccgcgctgcgaggcgcccgccgcggcacccgaggtctcggaggagccgcggtgcccgcgcgccgcctgccaggccaagcgcggggaccagcgctgcgaccgcgagtgcaacagcccaggctgcggctgggacggcggcgactgctcgctgagcgtgggcgacccctggcggcaatgcgaggcgctgcagtgctggcgcctcttcaacaacagccgctgcgaccccgcctgcagctcgcccgcctgcctctacgacaacttcgactgccacgccggtggccgcgagcgcacttgcaacccggtgtacgagaagtactgcgccgaccactttgccgacggccgctgcgaccagggctgcaacacggaggagtgcggctgggatgggctggattgtgccagcgaggtgccggccctgctggcccgcggcgtgctggtgctcacagtgctgctgccgccagaggagctactgcgttccagcgccgactttctgcagcggctcagcgccatcctgcgcacctcgctgcgcttccgcctggacgcgcacggccaggccatggtcttcccttaccaccggcctagtcctggctccgaaccccgggcccgtcgggagctggcccccgaggtgatcggctcggtagtaatgctggagattgacaaccggctctgcctgcagtcgcctgagaatgatcactgcttccccgatgcccagagcgccgctgactacctgggagcgttgtcagcggtggagcgcctggacttcccgtacccactgcgggacgtgcggggggagccgctggagcctccagaacccagcgtcccgctgctgccactgctagtggcgggcgctgtcttgctgctggtcattctcgtcctgggtgtcatggtggcccggcgcaagcgcgagcacagcaccctctggttccctgagggcttctcactgcacaaggacgtggcctctggtcacaagggccggcgggaacccgtgggccaggacgcgctgggcatgaagaacatggccaagggtgagagcctgatgggggaggtggccacagactggatggacacagagtgcccagaggccaagcggctaaaggtagaggagccaggcatgggggctgaggaggctgtggattgccgtcagtggactcaacaccatctggttgctgctgacatccgcgtggcaccagccatggcactgacaccaccacagggcgacgcagatgctgatggcatggatgtcaatgtgcgtggcccagatggcttcaccccgctaatgctggcttccttctgtgggggggctctggagccaatgccaactgaagaggatgaggcagatgacacatcagctagcatcatctccgacctgatctgccagggggctcagcttggggcacggactgaccgtactggcgagactgctttgcacctggctgcccgttatgcccgtgctgatgcagccaagcggctgctggatgctggggcagacaccaatgcccaggaccactcaggccgcactcccctgcacacagctgtcacagccgatgcccagggtgtcttccagattctcatccgaaaccgctctacagacttggatgcccgcatggcagatggctcaacggcactgatcctggcggcccgcctggcagtagagggcatggtggaagagctcatcgccagccatgctgatgtcaatgctgtggatgagcttgggaaatcagccttacactgggctgcggctgtgaacaacgtggaagccactttggccctgctcaaaaatggagccaataaggacatgcaggatagcaaggaggagacccccctattcctggccgcccgcgagggcagctatgaggctgccaagctgctgttggaccactttgccaaccgtgagatcaccgaccacctggacaggctgccgcgggacgtagcccaggagagactgcaccaggacatcgtgcgcttgctggatcaacccagtgggccccgcagcccccccggtccccacggcctggggcctctgctctgtcctccaggggccttcctccctggcctcaaagcggcacagtcggggtccaagaagagcaggaggccccccgggaaggcggggctggggccgcaggggccccgggggcggggcaagaagctgacgctggcctgcccgggccccctggctgacagctcggtcacgctgtcgcccgtggactcgctggactccccgcggcctttcggtgggccccctgcttcccctggtggcttcccccttgaggggccctatgcagctgccactgccactgcagtgtctctggcacagcttggtggcccaggccgggcgggtctagggcgccagccccctggaggatgtgtactcagcctgggcctgctgaaccctgtggctgtgcccctcgattgggcccggctgcccccacctgcccctccaggcccctcgttcctgctgccactggcgccgggaccccagctgctcaacccagggacccccgtctccccgcaggagcggcccccgccttacctggcagtcccaggacatggcgaggagtacccggcggctggggcacacagcagccccccaaaggcccgcttcctgcgggttcccagtgagcacccttacctgaccccatcccccgaatcccctgagcactgggccagcccctcacctccctccctctcagactggtccgaatccacgcctagcccagccactgccactggggccatggccaccaccactggggcactgcctgcccagccacttcccttgtctgttcccagctcccttgctcaggcccagacccagctggggccccagccggaagttacccccaagaggcaagtgttggcctga. (SEQ ID NO: 74)Nurr1: NM_006186: atgccttgtgttcaggcgcagtatgggtcctcgcctcaaggagccagccccgcttctcagagctacagttaccactcttcgggagaatacagctccgatttcttaactccagagtttgtcaagtttagcatggacctcaccaacactgaaatcactgccaccacttctctccccagcttcagtacctttatggacaactacagcacaggctacgacgtcaagccaccttgcttgtaccaaatgcccctgtccggacagcagtcctccattaaggtagaagacattcagatgcacaactaccagcaacacagccacctgcccccccagtctgaggagatgatgccgcactccgggtcggtttactacaagccctcctcgcccccgacgcccaccaccccgggcttccaggtgcagcacagccccatgtgggacgacccgggatctctccacaacttccaccagaactacgtggccactacgcacatgatcgagcagaggaaaacgccagtctcccgcctctccctcttctcctttaagcaatcgccccctggcaccccggtgtctagttgccagatgcgcttcgacgggcccctgcacgtccccatgaacccggagcccgccggcagccaccacgtggtggacgggcagaccttcgctgtgcccaaccccattcgcaagcccgcgtccatgggcttcccgggcctgcagatcggccacgcgtctcagctgctcgacacgcaggtgccctcaccgccgtcgcggggctccccctccaacgaggggctgtgcgctgtgtgtggggacaacgcggcctgccaacactacggcgtgcgcacctgtgagggctgcaaaggcttctttaagcgcacagtgcaaaaaaatgcaaaatacgtgtgtttagcaaataaaaactgcccagtggacaagcgtcgccggaatcgctgtcagtactgccgatttcagaagtgcctggctgttgggatggtcaaagaagtggttcgcacagacagtttaaaaggccggagaggtcgtttgccctcgaaaccgaagagcccacaggagccctctcccccttcgcccccggtgagtctgatcagtgccctcgtcagggcccatgtcgactccaacccggctatgaccagcctggactattccaggttccaggcgaaccctgactatcaaatgagtggagatgacacccagcatatccagcaattctatgatctcctgactggctccatggagatcatccggggctgggcagagaagatccctggcttcgcagacctgcccaaagccgaccaagacctgctttttgaatcagctttcttagaactgtttgtccttcgattagcatacaggtccaacccagtggagggtaaactcatcttttgcaatggggtggtcttgcacaggttgcaatgcgttcgtggctttggggaatggattgattccattgttgaattctcctccaacttgcagaatatgaacatcgacatttctgccttctcctgcattgctgccctggctatggtcacagagagacacgggctcaaggaacccaagagagtggaagaactgcaaaacaagattgtaaattgtctcaaagaccacgtgactttcaacaatggggggttgaaccgccccaattatttgtccaaactgttggggaagctcccagaacttcgtaccctttgcacacaggggctacagcgcattttctacctgaaattggaagacttggtgccaccgccagcaataattgacaaacttttcctggacactttacctttctaa; NMO19328. (SEQ ID NO: 75)NOV(CCN3): NM_002514: atgcagagtgtgcagagcacgagcttttgtctccgaaagcagtgcctttgcctgaccttcctgcttctccatctcctgggacaggtcgctgcgactcagcgctgccctccccagtgcccgggccggtgccctgcgacgccgccgacctgcgcccccggggtgcgcgcggtgctggacggctgctcatgctgtctggtgtgtgcccgccagcgtggcgagagctgctcagatctggagccatgcgacgagagcagtggcctctactgtgatcgcagcgcggaccccagcaaccagactggcatctgcacggcggtagagggagataactgtgtgttcgatggggtcatctaccgcagtggagagaaatttcagccaagctgcaaattccagtgcacctgcagagatgggcagattggctgtgtgccccgctgtcagctggatgtgctactgcctgagcctaactgcccagctccaagaaaagttgaggtgcctggagagtgctgtgaaaagtggatctgtggcccagatgaggaggattcactgggaggccttacccttgcagcttacaggccagaagccaccctaggagtagaagtctctgactcaagtgtcaactgcattgaacagaccacagagtggacagcatgctccaagagctgtggtatggggttctccacccgggtcaccaataggaaccgtcaatgtgagatgctgaaacagactcggctctgcatggtgcggccctgtgaacaagagccagagcagccaacagataagaaaggaaaaaagtgtctccgcaccaagaagtcactcaaagccatccacctgcagttcaagaactgcaccagcctgcacacctacaagcccaggttctgtggggtctgcagtgatggccgctgctgcactccccacaataccaaaaccatccaggcagagtttcagtgctccccagggcaaatagtcaagaagccagtgatggtcattgggacctgcacctgtcacaccaactgtcctaagaacaatgaggccttcctccaggagctggagctgaagactaccagagggaaaatgtaa; NM_010930; NM030868; BCO15028.(SEQ ID NO: 76)OLIG1:NM_138983: atgctgcggccacagcggcccggagacttgcagctcggggcctccctctacgagctggtgggctacaggcagccgccctcctcctcctcctcctccacctcctccacctcctccacttcctcctcctccacgacggcccccctcctccccaaggctgcgcgcgagaagccggaggcgccggccgagcctccaggccccgggcccgggtcaggcgcgcacccgggcggcagcgcccggccggacgccaaggaggagcagcagcagcagctgcggcgcaagatcaacagccgcgagcggaagcgcatgcaggacctgaacctggccatggacgccctgcgcgaggtcatcctgccctactcagcggcgcactgccagggcgcgcccggccgcaagctctccaagatagccacgctgctgctcgcccgcaactacatcctactgctgggcagctcgctgcaggagctgcgccgcgcgctgggcgagggcgccgggcccgccgcgccgcgcctgctgctggccgggctgcccctgctcgccgccgcgcccggctccgtgttgctggcgcccggcgccgtaggaccccccgacgcgctgcgccccgccaagtacctgtcgctggcgctggacgagccgccgtgcggccagttcgctctccccggcggcggcgcaggcggccccggcctctgcacctgcgccgtgtgcaagttcccgcacctggtcccggccagcctgggcctggccgccgtgcaggcgcaattctccaagtga; NM016968; NMJ)01020796. (SEQ ID NO: 77) OLIGiNM{circumflex over( )}OSδOóiatggactcggacgccagcctggtgtccagccgcccgtcgtcgccagagcccgatgacctttttctgccggcccggagtaagggcagcagcggcagcgccttcactgggggcaccgtgtcctcgtccaccccgagtgactgcccgccggagctgagcgccgagctgcgcggcgctatgggctctgcgggcgcgcatcctggggacaagctaggaggcagtggcttcaagtcatcctcgtccagcacctcgtcgtctacgtcgtcggcggctgcgtcgtccaccaagaaggacaagaagcaaatgacagagccggagctgcagcagctgcgtctcaagatcaacagccgcgagcgcaagcgcatgcacgacctcaacatcgccatggatggcctccgcgaggtcatgccgtacgcacacggcccttcggtgcgcaagctttccaagatcgccacgctgctgctggcgcgcaactacatcctcatgctcaccaactcgctggaggagatgaagcgactggtgagcgagatctacgggggccaccacgctggcttccacccgtcggcctgcggcggcctggcgcactccgcgcccctgcccgccgccaccgcgcacccggcagcagcagcgcacgccgcacatcaccccgcggtgcaccaccccatcctgccgcccgccgccgcagcggctgctgccgccgctgcagccgcggctgtgtccagcgcctctctgcccggatccgggctgccgtcggtcggctccatccgtccaccgcacggcctactcaagtctccgtctgctgccgcggccgccccgctggggggcgggggcggcggcagtggggcgagcgggggcttccagcactggggcggcatgccctgcccctgcagcatgtgccaggtgccgccgccgcaccaccacgtgtcggctatgggcgccggcagcctgccgcgcctcacctccgacgccaagtga. (SEQ ID NO: 78)PcIxI: NM000209: atgaacggcgaggagcagtactacgcggccacgcagctttacaaggacccatgcgcgttccagcgaggcccggcgccggagttcagcgccagcccccctgcgtgcctgtacatgggccgccagcccccgccgccgccgccgcacccgttccctggcgccctgggcgcgctggagcagggcagccccccggacatctccccgtacgaggtgccccccctcgccgacgaccccgcggtggcgcaccttcaccaccacctcccggctcagctcgcgctcccccacccgcccgccgggcccttcccggagggagccgagccgggcgtcctggaggagcccaaccgcgtccagctgcctttcccatggatgaagtctaccaaagctcacgcgtggaaaggccagtgggcaggcggcgcctacgctgcggagccggaggagaacaagcggacgcgcacggcctacacgcgcgcacagctgctagagctggagaaggagttcctattcaacaagtacatctcacggccgcgccgggtggagctggctgtcatgttgaacttgaccgagagacacatcaagatctggttccaaaaccgccgcatgaagtggaaaaaggaggaggacaagaagcgcggcggcgggacagctgtcgggggtggcggggtcgcggagcctgagcaggactgcgccgtgacctccggcgaggagcttctggcgctgccgccgccgccgccccccggaggtgctgtgccgcccgctgcccccgttgccgcccgagagggccgcctgccgcctggccttagcgcgtcgccacagccctccagcgtcgcgcctcggcggccgcaggaaccacgatga; NM008814; NM022852. (SEQ ID NO: 79)Pet1(FEV):BC138435; NM017521: atgagacagagcggcgcctcccagcccctgctgatcaacatgtacctgccagatcccgtcggagacggtctcttcaaggacgggaagaacccgagctgggggccgctgagccccgcggttcagaaaggcagcggacagatccagctgtggcagtttctgctggagctgctggctgaccgcgcgaacgccggctgcatcgcgtgggagggcggtcacggcgagttcaagctcacggacccggacgaggtggcgcggcggtggggcgagcgcaagagcaagcccaacatgaactacgacaagctgagccgcgccctgcgctactactacgacaagaacatcatgagcaaggtgcatggcaagcgctacgcctaccgcttcgacttccagggcctggcgcaggcctgccagccgccgcccgcgcacgctcatgccgccgccgcagctgctgccgccgccgcggccgcccaggacggcgcgctctacaagctgcccgccggcctcgccccgctgcccttccccggcctctccaaactcaacctcatggccgcctcggccggggtcgcgcccgccggcttctcctactggccgggcccgggccccgccgccaccgctgccgccgccaccgccgcgctctaccccagtcccagcttgcagcccccgcccgggcccttcggggccgtggccgcagcctcgcacttggggggccattaccactag; NG_002690; NP059991.(SEQ ID NO: 80)Phox2a: NM_005169: atggactactcctacctcaattcgtacgactcgtgcgtggcggccatggaggcgtccgcctacggcgactttggcgcctgcagccagcccggcggcttccaatacagccccctgcggcccgctttccccgcggcagggccgccctgccccgcgctcggctcctccaactgcgcacttggcgccctacgcgaccaccagcccgcgccctactcggcagtgccctacaagttcttcccagagccatccggcctgcacgagaagcgcaagcagcggcgcatccgcaccacgttcaccagcgcgcagctcaaggagctggagcgcgttttcgctgagacccactaccccgacatttacacgcgtgaggagctggcgctcaagatcgacctcactgaggctcgcgtgcaggtctggttccagaaccgccgggccaagttccgcaaacaggagcgcgcggccagcgccaagggcgcggcgggcgcggcgggcgccaaaaagggcgaggcgcgctgctcctccgaggacgacgattccaaggagtccacgtgcagccccacgcccgatagcaccgcctcgctgccgccgccgcctgcgcccggcctggccagcccgcgcctgagccccagcccgctgcccgtcgcactgggctccgggccgggacctgggccggggccacagccgctcaagggcgcactgtgggccggtgtggcgggcggtgggggcggcgggcctggcgcgggagcggccgaactacttaaggcttggcagccggcggagtccggccccgggcccttctccggggttctgtcctcctttcaccggaagcccggccccgccctgaagaccaatctcttctag; AJ320270; AY371497; AY371496. (SEQ ID NO: 81)Phox2b: NM_003924: atgtataaaatggaatattcttacctcaattcctctgcctacgagtcctgtatggctgggatggacacctcgagcctggcttcagcctatgctgacttcagttcctgcagccaggccagtggcttccagtataacccgataaggaccacttttggggccacgtccggctgcccttccctcacgccgggatcctgcagcctgggcaccctcagggaccaccagagcagtccgtacgccgcagttccttacaaactcttcacggaccacggcggcctcaacgagaagcgcaagcagcggcgcatccgcaccactttcaccagtgcccagctcaaagagctggaaagggtcttcgcggagactcactaccccgacatctacactcgggaggagctggccctgaagatcgacctcacagaggcgcgagtccaggtgtggttccagaaccgccgcgccaagtttcgcaagcaggagcgcgcagcggcagccgcagcggccgcggccaagaacggctcctcgggcaaaaagtctgactcttccagggacgacgagagcaaagaggccaagagcactgacccggacagcactgggggcccaggtcccaatcccaaccccacccccagctgcggggcgaatggaggcggcggcggcgggcccagcccggctggagctccgggggcggcggggcccgggggcccgggaggcgaacccggcaagggcggcgcagcagcagcggcggcggccgcggcagcggcggcggcggcagcggcagcggcggcagctggaggcctggctgcggctgggggccctggacaaggctgggctcccggccccggccccatcacctccatcccggattcgcttgggggtcccttcgccagcgtcctatcttcgctccaaagacccaacggtgccaaagccgccttagtgaagagcagtatgttctga; NM008888; AY371498; Y14493. (SEQ ID NO: 82)Pit1: NM_000306: atgagttgccaagcttttacttcggctgatacctttatacctctgaattctgacgcctctgcaactctgcctctgataatgcatcacagtgctgccgagtgtctaccagtctccaaccatgccaccaatgtgatgtctacagcaacaggacttcattattctgttccttcctgtcattatggaaaccagccatcaacctatggagtgatggcaggtagtttaaccccttgtctttataaatttcctgaccacaccttgagtcatggatttcctcctatacaccagcctcttctggcagaggaccccacagctgctgatttcaagcaggaactcaggcggaaaagtaaattggtggaagagccaatagacatggattctccagaaatcagagaacttgaaaagtttgccaatgaatttaaagtgagacgaattaaattaggatacacccagacaaatgttggggaggccctggcagctgtgcatggctctgaattcagtcaaacaacaatctgccgatttgaaaatctgcagctcagctttaaaaatgcatgcaaactgaaagcaatattatccaaatggctggaggaagctgagcaagtaggagctttgtacaatgaaaaagtgggagcaaatgaaaggaaaagaaaacgaagaacaactataagcattgctgctaaagatgctctggagagacactttggagaacagaataaaccttcttctcaagagatcatgaggatggctgaagaactgaatctggagaaagaagtagtaagagtttggttttgcaaccggaggcagagagaaaaacgggtgaaaacaagtctgaatcagagtttattttctatttctaaggaacatcttgagtgcagataa; M23253.(SEQ ID NO: 83)PITX3: NM_005029: atggagttcggcctgctcagcgaggcagaggcccggagccctgccctgtcgctgtcagacgctggcactccgcacccccagctcccagagcacggctgcaagggccaggagcacagcgactcagaaaaggcctcggcttcgctgcccggcggctccccagaggacggttcgctgaaaaagaagcagcggcggcagcgcacgcacttcaccagccagcagctacaggagctagaggcgaccttccagaggaaccgctaccccgacatgagcacgcgcgaggagatcgccgtgtggaccaacctcaccgaggcccgcgtgcgggtgtggttcaagaaccggcgcgccaaatggcggaagcgcgagcgcagccagcaggccgagctatgcaaaggcagcttcgcggcgccgctcggggggctggtgccgccctacgaggaggtgtaccccggctactcgtacggcaactggccgcccaaggctcttgccccgccgctcgccgccaagacctttccattcgccttcaactcggtcaacgtggggcctctggcttcgcagcccgtcttctcgccacccagctccatcgccgcctccatggtgccctccgccgcggctgccccgggcaccgtgccagggcctggggccctgcagggcctgggcgggggcccccccgggctggctccggccgccgtgtcctccggggccgtgtcctgcccttatgcctcggccgccgccgccgccgcggctgccgcctcttccccctacgtctatcgggacccgtgtaactcgagcctggccagcctgcggctcaaagccaaacagcacgcctccttcagctaccccgctgtgcacgggccgcccccggcagccaaccttagtccgtgccagtacgccgtggaaaggcccgtatga; NM008852; NM008987;(SEQ ID NO: 84)RUNX1: NM001001890: atgcgtatccccgtagatgccagcacgagccgccgcttcacgccgccttccaccgcgctgagcccaggcaagatgagcgaggcgttgccgctgggcgccccggacgccggcgctgccctggccggcaagctgaggagcggcgaccgcagcatggtggaggtgctggccgaccacccgggcgagctggtgcgcaccgacagccccaacttcctctgctccgtgctgcctacgcactggcgctgcaacaagaccctgcccatcgctttcaaggtggtggccctaggggatgttccagatggcactctggtcactgtgatggctggcaatgatgaaaactactcggctgagctgagaaatgctaccgcagccatgaagaaccaggttgcaagatttaatgacctcaggtttgtcggtcgaagtggaagagggaaaagcttcactctgaccatcactgtcttcacaaacccaccgcaagtcgccacctaccacagagccatcaaaatcacagtggatgggccccgagaacctcgaagacatcggcagaaactagatgatcagaccaagcccgggagcttgtccttttccgagcggctcagtgaactggagcagctgcggcgcacagccatgagggtcagcccacaccacccagcccccacgcccaaccctcgtgcctccctgaaccactccactgcctttaaccctcagcctcagagtcagatgcaggatacaaggcagatccaaccatccccaccgtggtcctacgatcagtcctaccaatacctgggatccattgcctctccttctgtgcacccagcaacgcccatttcacctggacgtgccagcggcatgacaaccctctctgcagaactttccagtcgactctcaacggcacccgacctgacagcgttcagcgacccgcgccagttccccgcgctgccctccatctccgacccccgcatgcactatccaggcgccttcacctactccccgacgccggtcacctcgggcatcggcatcggcatgtcggccatgggctcggccacgcgctaccacacctacctgccgccgccctaccccggctcgtcgcaagcgcagggaggcccgttccaagccagctcgccctcctaccacctgtactacggcgcctcggccggctcctaccagttctccatggtgggcggcgagcgctcgccgccgcgcatcctgccgccctgcaccaacgcctccaccggctccgcgctgctcaaccccagcctcccgaaccagagcgacgtggtggaggccgagggcagccacagcaactcccccaccaacatggcgccctccgcgcgcctggaggaggccgtgtggaggccctactga; AY509916; AY509915; NM_001001890.2; NP001001890.1;NM001122607.1; NP001116079.1; NM001754.4; NPJ)01745.2 (SEQ ID NO: 85)Runx2: NMJ)01015051: atgcttcattcgcctcacaaacaaccacagaaccacaagtgcggtgcaaactttctccaggaggacagcaagaagtctctggtttttaaatggttaatctccgcaggtcactaccagccaccgagaccaacagagtcatttaaggctgcaagcagtatttacaacagagggtacaagttctatctgaaaaaaaaaggagggactatggcatcaaacagcctcttcagcacagtgacaccatgtcagcaaaacttcttttgggatccgagcaccagccggcgcttcagccccccctccagcagcctgcagcccggcaaaatgagcgacgtgagcccggtggtggctgcgcaacagcagcagcaacagcagcagcagcaacagcagcagcagcagcagcaacagcagcagcagcagcaggaggcggcggcggcggctgcggcggcggcggcggctgcggcggcggcagctgcagtgccccggttgcggccgccccacgacaaccgcaccatggtggagatcatcgccgaccacccggccgaactcgtccgcaccgacagccccaacttcctgtgctcggtgctgccctcgcactggcgctgcaacaagaccctgcccgtggccttcaaggtggtagccctcggagaggtaccagatgggactgtggttactgtcatggcgggtaacgatgaaaattattctgctgagctccggaatgcctctgctgttatgaaaaaccaagtagcaaggttcaacgatctgagatttgtgggccggagtggacgaggcaagagtttcaccttgaccataaccgtcttcacaaatcctccccaagtagctacctatcacagagcaattaaagttacagtagatggacctcgggaacccagaaggcacagacagaagcttgatgactctaaacctagtttgttctctgaccgcctcagtgatttagggcgcattcctcatcccagtatgagagtaggtgtcccgcctcagaacccacggccctccctgaactctgcaccaagtccttttaatccacaaggacagagtcagattacagaccccaggcaggcacagtcttccccgccgtggtcctatgaccagtcttacccctcctacctgagccagatgacgtccccgtccatccactctaccaccccgctgtcttccacacggggcactgggcttcctgccatcaccgatgtgcctaggcgcatttcaggtgcttcagaactgggccctttttcagaccccaggcagttcccaagcatttcatccctcactgagagccgcttctccaacccacgaatgcactatccagccacctttacttacaccccgccagtcacctcaggcatgtccctcggtatgtccgccaccactcactaccacacctacctgccaccaccctaccccggctcttcccaaagccagagtggacccttccagaccagcagcactccatatctctactatggcacttcgtcaggatcctatcagtttcccatggtgccggggggagaccggtctccttccagaatgcttccgccatgcaccaccacctcgaatggcagcacgctattaaatccaaatttgcctaaccagaatgatggtgttgacgctgatggaagccacagcagttccccaactgttttgaattctagtggcagaatggatgaatctgtttggcgaccatattga; NM001015051.2; NP001015051.2; NM001015051; NM001024630.2;NP001019801.2; NM004348.3; NP004339.3. (SEQ ID NO: 86)Shh: NM_000193: atgctgctgctggcgagatgtctgctgctagtcctcgtctcctcgctgctggtatgctcgggactggcgtgcggaccgggcagggggttcgggaagaggaggcaccccaaaaagctgacccctttagcctacaagcagtttatccccaatgtggccgagaagaccctaggcgccagcggaaggtatgaagggaagatctccagaaactccgagcgatttaaggaactcacccccaattacaaccccgacatcatatttaaggatgaagaaaacaccggagcggacaggctgatgactcagaggtgtaaggacaagttgaacgctttggccatctcggtgatgaaccagtggccaggagtgaaactgcgggtgaccgagggctgggacgaagatggccaccactcagaggagtctctgcactacgagggccgcgcagtggacatcaccacgtctgaccgcgaccgcagcaagtacggcatgctggcccgcctggcggtggaggccggcttcgactgggtgtactacgagtccaaggcacatatccactgctcggtgaaagcagagaactcggtggcggccaaatcgggaggctgcttcccgggctcggccacggtgcacctggagcagggcggcaccaagctggtgaaggacctgagccccggggaccgcgtgctggcggcggacgaccagggccggctgctctacagcgacttcctcactttcctggaccgcgacgacggcgccaagaaggtcttctacgtgatcgagacgcgggagccgcgcgagcgcctgctgctcaccgccgcgcacctgctctttgtggcgccgcacaacgactcggccaccggggagcccgaggcgtcctcgggctcggggccgccttccgggggcgcactggggcctcgggcgctgttcgccagccgcgtgcgcccgggccagcgcgtgtacgtggtggccgagcgtgacggggaccgccggctcctgcccgccgctgtgcacagcgtgaccctaagcgaggaggccgcgggcgcctacgcgccgctcacggcccagggcaccattctcatcaaccgggtgctggcctcgtgctacgcggtcatcgaggagcacagctgggcgcaccgggccttcgcgcccttccgcctggcgcacgcgctcctggctgcactggcgcccgcgcgcacggaccgcggcggggacagcggcggcggggaccgcgggggcggcggcggcagagtagccctaaccgctccaggtgctgccgacgctccgggtgcgggggccaccgcgggcatccactggtactcgcagctgctctaccaaataggcacctggctcctggacagcgaggccctgcacccgctgggcatggcggtcaagtccagctga;NP000184; NM_009170; NP033196; NM20482]; NP_990152. (SEQ ID NO: 87)Sox9: NM_000346: atgaatctcctggaccccttcatgaagatgaccgacgagcaggagaagggcctgtccggcgcccccagccccaccatgtccgaggactccgcgggctcgccctgcccgtcgggctccggctcggacaccgagaacacgcggccccaggagaacacgttccccaagggcgagcccgatctgaagaaggagagcgaggaggacaagttccccgtgtgcatccgcgaggcggtcagccaggtgctcaaaggctacgactggacgctggtgcccatgccggtgcgcgtcaacggctccagcaagaacaagccgcacgtcaagcggcccatgaacgccttcatggtgtgggcgcaggcggcgcgcaggaagctcgcggaccagtacccgcacttgcacaacgccgagctcagcaagacgctgggcaagctctggagacttctgaacgagagcgagaagcggcccttcgtggaggaggcggagcggctgcgcgtgcagcacaagaaggaccacccggattacaagtaccagccgcggcggaggaagtcggtgaagaacgggcaggcggaggcagaggaggccacggagcagacgcacatctcccccaacgccatcttcaaggcgctgcaggccgactcgccacactcctcctccggcatgagcgaggtgcactcccccggcgagcactcggggcaatcccagggcccaccgaccccacccaccacccccaaaaccgacgtgcagccgggcaaggctgacctgaagcgagaggggcgccccttgccagaggggggcagacagccccctatcgacttccgcgacgtggacatcggcgagctgagcagcgacgtcatctccaacatcgagaccttcgatgtcaacgagtttgaccagtacctgccgcccaacggccacccgggggtgccggccacgcacggccaggtcacctacacgggcagctacggcatcagcagcaccgcggccaccccggcgagcgcgggccacgtgtggatgtccaagcagcaggcgccgccgccacccccgcagcagcccccacaggccccgccggccccgcaggcgcccccgcagccgcaggcggcgcccccacagcagccggcggcacccccgcagcagccacaggcgcacacgctgaccacgctgagcagcgagccgggccagtcccagcgaacgcacatcaagacggagcagctgagccccagccactacagcgagcagcagcagcactcgccccaacagatcgcctacagccccttcaacctcccacactacagcccctcctacccgcccatcacccgctcacagtacgactacaccgaccaccagaactccagctcctactacagccacgcggcaggccagggcaccggcctctactccaccttcacctacatgaaccccgctcagcgccccatgtacacccccatcgccgacacctctggggtcccttccatcccgcagacccacagcccccagcactgggaacaacccgtctacacacagctcactcgaccttgaNM000346; NP000337; NM011448; NP035578; XM_343981; XP_343982.(SEQ ID NO: 88)sox17: NM_022454: atgagcagcccggatgcgggatacgccagtgacgaccagagccagacccagagcgcgctgcccgcggtgatggccgggctgggcccctgcccctgggccgagtcgctgagccccatcggggacatgaaggtgaagggcgaggcgccggcgaacagcggagcaccggccggggccgcgggccgagccaagggcgagtcccgtatccggcggccgatgaacgctttcatggtgtgggctaaggacgagcgcaagcggctggcgcagcagaatccagacctgcacaacgccgagttgagcaagatgctgggcaagtcgtggaaggcgctgacgctggcggagaagcggcccttcgtggaggaggcagagcggctgcgcgtgcagcacatgcaggaccaccccaactacaagtaccggccgcggcggcgcaagcaggtgaagcggctgaagcgggtggagggcggcttcctgcacggcctggctgagccgcaggcggccgcgctgggccccgagggcggccgcgtggccatggacggcctgggcctccagttccccgagcagggcttccccgccggcccgccgctgctgcctccgcacatgggcggccactaccgcgactgccagagtctgggcgcgcctccgctcgacggctacccgttgcccacgcccgacacgtccccgctggacggcgtggaccccgacccggctttcttcgccgccccgatgcccggggactgcccggcggccggcacctacagctacgcgcaggtctcggactacgctggccccccggagcctcccgccggtcccatgcacccccgactcggcccagagcccgcgggtccctcgattccgggcctcctggcgccacccagcgcccttcacgtgtactacggcgcgatgggctcgcccggggcgggcggcgggcgcggcttccagatgcagccgcaacaccagcaccagcaccagcaccagcaccaccccccgggccccggacagccgtcgccccctccggaggcactgccctgccgggacggcacggaccccagtcagcccgccgagctcctcggggaggtggaccgcacggaatttgaacagtatctgcacttcgtgtgcaagcctgagatgggcctcccctaccaggggcatgactccggtgtgaatctccccgacagccacggggccatttcctcggtggtgtccgacgccagctccgcggtatattactgcaactatcctgacgtgtga; BC140307; NM_011441. (SEQ ID NO: 89)DLX2: NM004405: atgactggagtctttgacagtctagtggctgatatgcactcgacccagatcgccgcctccagcacgtaccaccagcaccagcagcccccgagcggcggcggcgccggcccgggtggcaacagcagcagcagcagcagcctccacaagccccaggagtcgcccacccttccggtgtccaccgccaccgacagcagctactacaccaaccagcagcacccggcgggcggcggcggcggcgggggctcgccctacgcgcacatgggttcctaccagtaccaagccagcggcctcaacaacgtcccttactccgccaagagcagctatgacctgggctacaccgccgcctacacctcctacgctccctatggaaccagttcgtccccagccaacaacgagcctgagaaggaggaccttgagcctgaaattcggatagtgaacgggaagccaaagaaagtccggaaaccccgcaccatctactccagtttccagctggcggctcttcagcggcgtttccaaaagactcaatacttggccttgccggagcgagccgagctggcggcctctctgggcctcacccagactcaggtcaaaatctggttccagaaccgccggtccaagttcaagaagatgtggaaaagtggtgagatcccctcggagcagcaccctggggccagcgcttctccaccttgtgcttcgccgccagtctcagcgccggcctcctgggactttggtgtgccgcagcggatggcgggcggcggtggtccgggcagtggcggcagcggcgccggcagctcgggctccagcccgagcagcgcggcctcggcttttctgggcaactacccctggtaccaccagacctcgggatccgcctcacacctgcaggccacggcgccgctgctgcaccccactcagaccccgcagccgcatcaccaccaccaccatcacggcggcgggggcgccccggtgagcgcggggacgattttctaa; NP_004396.1; NM010054.(SEQ ID NO: 90)DLX5: NM_005221: atgacaggagtgtttgacagaagggtccccagcatccgatccggcgacttccaagctccgttccagacgtccgcagctatgcaccatccgtctcaggaatcgccaactttgcccgagtcttcagctaccgattctgactactacagccctacggggggagccccgcacggctactgctctcctacctcggcttcctatggcaaagctctcaacccctaccagtatcagtatcacggcgtgaacggctccgccgggagctacccagccaaagcttatgccgactatagctacgctagctcctaccaccagtacggcggcgcctacaaccgcgtcccaagcgccaccaaccagccagagaaagaagtgaccgagcccgaggtgagaatggtgaatggcaaaccaaagaaagttcgtaaacccaggactatttattccagctttcagctggccgcattacagagaaggtttcagaagactcagtacctcgccttgccggaacgcgccgagctggccgcctcgctgggattgacacaaacacaggtgaaaatctggtttcagaacaaaagatccaagatcaagaagatcatgaaaaacggggagatgcccccggagcacagtcccagctccagcgacccaatggcgtgtaactcgccgcagtctccagcggtgtgggagccccagggctcgtcccgctcgctcagccaccaccctcatgcccaccctccgacctccaaccagtccccagcgtccagctacctggagaactctgcatcctggtacacaagtgcagccagctcaatcaattcccacctgccgccgccgggctccttacagcacccgctggcgctggcctccgggacactctattag;;NM005221; NP005212. (SEQ ID NO: 91)HES1: NM_005524: atgccagctgatataatggagaaaaattcctcgtccccggtggctgctaccccagccagtgtcaacacgacaccggataaaccaaagacagcatctgagcacagaaagtcatcaaagcctattatggagaaaagacgaagagcaagaataaatgaaagtctgagccagctgaaaacactgattttggatgctctgaagaaagatagctcgcggcattccaagctggagaaggcggacattctggaaatgacagtgaagcacctccggaacctgcagcgggcgcagatgacggctgcgctgagcacagacccaagtgtgctggggaagtaccgagccggcttcagcgagtgcatgaacgaggtgacccgcttcctgtccacgtgcgagggcgttaataccgaggtgcgcactcggctgctcggccacctggccaactgcatgacccagatcaatgccatgacctaccccgggcagccgcaccccgccttgcaggcgccgccaccgcccccaccgggacccggcggcccccagcacgcgccgttcgcgccgccgccgccactcgtgcccatccccgggggcgcggcgccccctcccggcggcgccccctgcaagctgggcagccaggctggagaggcggctaaggtgtttggaggcttccaggtggtaccggctcccgatggccagtttgctttcctcattcccaacggggccttcgcgcacagcggccctgtcatccccgtctacaccagcaacagcggcacctccgtgggccccaacgcagtgtcaccttccagcggcccctcgcttacggcggactccatgtggaggccgtggcggaactga;NP005515.1; NM008235; NP032261. (SEQ ID NO: 92)FGF8: NM_006119: atgggcagcccccgctccgcgctgagctgcctgctgttgcacttgctggtcctctgcctccaagcccaggtaactgttcagtcctcacctaattttacacagcatgtgagggagcagagcctggtgacggatcagctcagccgccgcctcatccggacctaccaactctacagccgcaccagcgggaagcacgtgcaggtcctggccaacaagcgcatcaacgccatggcagaggacggcgaccccttcgcaaagctcatcgtggagacggacacctttggaagcagagttcgagtccgaggagccgagacgggcctctacatctgcatgaacaagaaggggaagctgatcgccaagagcaacggcaaaggcaaggactgcgtcttcacggagattgtgctggagaacaactacacagcgctgcagaatgccaagtacgagggctggtacatggccttcacccgcaagggccggccccgcaagggctccaagacgcggcagcaccagcgtgaggtccacttcatgaagcggctgccccggggccaccacaccaccgagcagagcctgcgcttcgagttcctcaactacccgcccttcacgcgcagcctgcgcggcagccagaggacttgggcccccgagccccgatag; NM010205; NP034335; NM010205; NP034335; NP006110NM033163; NPJ49353; NM033164; NP149354; NM033165; NPJ49355.(SEQ ID NO: 93)PITX2: NM_000325: atgaactgcatgaaaggcccgcttcacttggagcaccgagcagcggggaccaagctgtcggccgtctcctcatcttcctgtcaccatccccagccgttagccatggcttcggttctggctcccggtcagccccggtcgctggactcctccaagcacaggctggaggtgcacaccatctccgacacctccagcccggaggccgcagagaaagataaaagccagcaggggaagaatgaggacgtgggcgccgaggacccgtctaagaagaagcggcaaaggcggcagcggactcactttaccagccagcagctccaggagctggaggccactttccagaggaaccgctacccggacatgtccacacgcgaagaaatcgctgtgtggaccaaccttacggaagcccgagtccgggtttggttcaagaatcgtcgggccaaatggagaaagagggagcgcaaccagcaggccgagctatgcaagaatggcttcgggccgcagttcaatgggctcatgcagccctacgacgacatgtacccaggctattcctacaacaactgggccgccaagggccttacatccgcctccctatccaccaagagcttccccttcttcaactctatgaacgtcaaccccctgtcatcacagagcatgttttccccacccaactctatctcgtccatgagcatgtcgtccagcatggtgccctcagcagtgacaggcgtcccgggctccagtctcaacagcctgaataacttgaacaacctgagtagcccgtcgctgaattccgcggtgccgacgcctgcctgtccttacgcgccgccgactcctccgtatgtttatagggacacgtgtaactcgagcctggccagcctgagactgaaagcaaagcagcactccagcttcggctacgccagcgtgcagaacccggcctccaacctgagtgcttgccagtatgcagtggaccggcccgtgtga; NM000325; NP000316; NM153426; NP700475; NMJ53427;NP700476; NM001042502; NP001035967; NM001042504; NP001035969.(SEQ ID NO: 94)REST4: DQ644039: atggccacccaggtgatggggcagtcttctggaggaggcagtctcttcaacaacagtgccaacatgggcatggccttaaccaacgacatgtacgacctgcacgagctctcgaaagctgaactggcagcccctcagctcatcatgttagccaacgtggccctgacgggggaggcaagcggcagctgctgcgattacctggtcggtgaagagaggcagatggccgaattgatgcccgtgggagacaaccacttctcagaaagtgaaggagaaggcctggaagagtcggctgacctcaaagggctggaaaacatggaactgggaagtttggagctaagtgctgtagaaccccagcccgtatttgaagcctcagctgccccagaaatatacagcgccaataaagatcccgctccagaaacacccgtggcggaagacaaatgcaggagttctaaggccaagcccttccggtgtaagccttgccagtacgaagccgaatctgaagagcagtttgtgcatcacatccggattcacagcgctaagaagttctttgtggaggaaagtgcagagaaacaggccaaagcctgggagtcggggtcgtctccggccgaagagggcgagttctccaaaggccccatccgctgtgaccgctgtggctacaataccaaccggtatgaccactacatggcacacctgaagcaccacctgcgagctggcgagaacgagcgcatctacaagtgcatcatctgcacgtacacgacggtcagcgagtaccactggaggaaacacctgagaaaccatttccccaggaaagtctacacctgcagcaagtgcaactacttctcagacagaaaaaataactacgttcagcacgtgcgaactcacacaggagaacgcccgtataaatgtgaactttgtccttactcaagctctcagaagactcatctaacgcgacacatgcggactcattcagagtgtgatctagctgggtga. (SEQ ID NO: 95)CREBbindingprotein: NM_134442: atgaccatggaatctggagccgagaaccagcagagtggagatgcagctgtaacagaagctgaaaaccaacaaatgacagttcaagcccagccacagattgccacattagcccaggtatctatgccagcagctcatgcaacatcatctgctcccaccgtaactctagtacagctgcccaatgggcagacagttcaagtccatggagtcattcaggcggcccagccatcagttattcagtctccacaagtccaaacagttcagtcttcctgtaaggacttaaaaagacttttctccggaacacagatttcaactattgcagaaagtgaagattcacaggagtcagtggatagtgtaactgattcccaaaagcgaagggaaattctttcaaggaggccttcctacaggaaaattttgaatgacttatcttctgatgcaccaggagtgccaaggattgaagaagagaagtctgaagaggagacttcagcacctgccatcaccactgtaacggtgccaactccaatttaccaaactagcagtggacagtatattgccattacccagggaggagcaatacagctggctaacaatggtaccgatggggtacagggcctgcaaacattaaccatgaccaatgcagcagccactcagccgggtactaccattctacagtatgcacagaccactgatggacagcagatcttagtgcccagcaaccaagttgttgttcaagctgcctctggagacgtacaaacataccagattcgcacagcacccactagcactattgcccctggagttgttatggcatcctccccagcacttcctacacagcctgctgaagaagcagcacgaaagagagaggtccgtctaatgaagaacagggaagcagctcgagagtgtcgtagaaagaagaaagaatatgtgaaatgtttagaaaacagagtggcagtgcttgaaaatcaaaacaagacattgattgaggagctaaaagcacttaaggacctttactgccacaaatcagatta;NM004379; NP004370; NP604391. (SEQ ID NO: 96) ZFp488: NM001013777:atggctgagggcaaaggggctcctctgaggccttcagttgagaagagatggaagctcatggaacccaagcagacccaggcagggatgttcaagaaaatgagccttgtggactctgacactgctgcaggaaagggtagccaagatgaggcctatactgaactgagcctgccaacagcaccgaacaagcctcgactggacaggcctcgggcctgcaaggcatacacagagcagaggcacaataccttcacagagctatcatgtctccaggagaggccaggggacatccaggcccagacgaggaagctggagaacccagaaggccagctcggccctcagcagctgccctcgagtttcctcagagcctcaggtgatggcacagtgtgttcagcatggccaggtgccccccggagtgagcagaaaagtgctttcagcaagccagccaaacgcccagcagagaaacctaagcgctctcccatgcttctggctggtggaagtgcagagggctcatgggagctctcaggactcatcaccactgtggacatcccatattgggctcatctgtcaactttcaagttcatgggtgatttctggaaattgcacacattgtcacagaacattctcctctgcaatgctttccagggggctcccacaccatggctggagcatacccaggtacaagcccccacatcctcagctccttcctccacagcctcccgggctctcttgccgcccacactctcctccttgggcttgtctactcagaactggtgtgcgaagtgcaacctagcctttcgcctgacagctgacctggtcttccacatgcggtcacatcacaaaagggaacacgtgggccctgacccacattctaagaaacgaagagaggaagttctcacttgccccgtttgccacgagtacttccgggagcgccaccatctgtccaggcatatggcttcacatagttag; BC089025;XM_224697; XP224697. (SEQ ID NO: 97)Foxa2: NM_021784: atgctgggagcggtgaagatggaagggcacgagccgtccgactggagcagctactatgcagagcccgagggctactcctccgtgagcaacatgaacgccggcctggggatgaacggcatgaacacgtacatgagcatgtcggcggccgccatgggcagcggctcgggcaacatgagcgcgggctccatgaacatgtcgtcgtacgtgggcgctggcatgagcccgtccctggcggggatgtcccccggcgcgggcgccatggcgggcatgggcggctcggccggggcggccggcgtggcgggcatggggccgcacttgagtcccagcctgagcccgctcggggggcaggcggccggggccatgggcggcctggccccctacgccaacatgaactccatgagccccatgtacgggcaggcgggcctgagccgcgcccgcgaccccaagacctacaggcgcagctacacgcacgcaaagccgccctactcgtacatctcgctcatcaccatggccatccagcagagccccaacaagatgctgacgctgagcgagatctaccagtggatcatggacctcttccccttctaccggcagaaccagcagcgctggcagaactccatccgccactcgctctccttcaacgactgtttcctgaaggtgccccgctcgcccgacaagcccggcaagggctccttctggaccctgcaccctgactcgggcaacatgttcgagaacggctgctacctgcgccgccagaagcgcttcaagtgcgagaagcagctggcgctgaaggaggccgcaggcgccgccggcagcggcaagaaggcggccgccggagcccaggcctcacaggctcaactcggggaggccgccgggccggcctccgagactccggcgggcaccgagtcgcctcactcgagcgcctccccgtgccaggagcacaagcgagggggcctgggagagctgaaggggacgccggctgcggcgctgagccccccagagccggcgccctctcccgggcagcagcagcaggccgcggcccacctgctgggcccgccccaccacccgggcctgccgcctgaggcccacctgaagccggaacaccactacgccttcaaccacccgttctccatcaacaacctcatgtcctcggagcagcagcaccaccacagccaccaccaccaccaaccccacaaaatggacctcaaggcctacgaacaggtgatgcactaccccggctacggttcccccatgcctggcagcttggccatgggcccggtcacgaacaaaacgggcctggacgcctcgcccctggccgcagatacctcctactaccagggggtgtactcccggcccattatgaactcctcttaa; NP_068556; NM_012743;NP036875; NMO10446; NP034576. Rnx (SEQ ID NO: 98)REN: NM_000537: atggatggatggagaaggatgcctcgctggggactgctgctgctgctctggggctcctgtacctttggtctcccgacagacaccaccacctttaaacggatcttcctcaagagaatgccctcaatccgagaaagcctgaaggaacgaggtgtggacatggccaggcttggtcccgagtggagccaacccatgaagaggctgacacttggcaacaccacctcctccgtgatcctcaccaactacatggacacccagtactatggcgagattgggatcgggaccccaccccaaaccttcaaagtcgtctttgacactggttcgtccaatgtttgggtgccctcctccaagtgcagccgtctctacactgcctgtgtgtatcacaagctcttcgatgcttcggattcctccagctacaagcacaatggaacagaactcaccctccgctattcaacagggacagtcagtggctttctcagccaggacatcatcaccgtgggtggaatcacggtgacacagatgtttggagaggtcacggagatgcccgccttacccttcatgctggccgagtttgatggggttgtgggcatgggcttcattgaacaggccattggcagggtcacccctatcttcgacaacatcatctcccaaggggtgctaaaagaggacgtcttctctttctactacaacagagattccgagaattcccaatcgctgggaggacagattgtgctgggaggcagcgacccccagcattacgaagggaatttccactatatcaacctcatcaagactggtgtctggcagattcaaatgaagggggtgtctgtggggtcatccaccttgctctgtgaagacggctgcctggcattggtagacaccggtgcatcctacatctcaggttctaccagctccatagagaagctcatggaggccttgggagccaagaagaggctgtttgattatgtcgtgaagtgtaacgagggccctacactccccgacatctattccacctgggaggcaaagaatacacgctcaccagcgcggactatgtatttcaggaatcctacagtagtaaaaagctgtgcacactggccatccacgccatggatatcccgccacccactggacccacctgggccctgggggccaccttcatccgaaagttctacacagagtttgatcggcgtaacaaccgcattggcttcgccttggcccgctga; (SEQ ID NO: 99) dL1AND(HAND2): NM_021973:atgagtctggtaggtggttttccccaccacccggtggtgcaccacgagggctacccgtttgccgccgccgccgccgcagctgccgccgccgccgccagccgctgcagccatgaggagaacccctacttccatggctggctcatcggccaccccgagatgtcgccccccgactacagcatggccctgtcctacagccccgagtatgccagcggcgccgccggcctggaccactcccattacgggggggtgccgccgggcgccgggcccccgggcctgggggggccgcgcccggtgaagcgccgaggcaccgccaaccgcaaggagcggcgcaggactcagagcatcaacagcgccttcgccgaactgcgcgagtgcatccccaacgtacccgccgacaccaaactctccaaaatcaagaccctgcgcctggccaccagctacatcgcctacctcatggacctgctggccaaggacgaccagaatggcgaggcggaggccttcaaggcagagatcaagaagaccgacgtgaaagaggagaagaggaagaaggagctgaacgaaatcttgaaaagcacagtgagcagcaacgacaagaaaaccaaaggccggacgggctggccgcagcacgtctgggccctggagctcaagcagtga; NMO10402; aspartoacylase (Canavan disease)(ASPA):NM_000049; (SEQ ID NO: 100)atgacttcttgtcacattgctgaagaacatatacaaaaggttgctatctttggaggaacccatgggaatgagctaaccggagtatltctggttaagcattggctagagaatggcgctgagattcagagaacagggctggaggtaaaaccatttattactaaccccagagcagtgaagaagtgtaccagatatattgactgtgacctgaatcgcatttttgaccttgaaaatcttggcaaaaaaatgtcagaagatttgccatatgaagtgagaagggctcaagaaataaatcatttatttggtccaaaagacagtgaagattcctatgacattatttttgaccttcacaacaccacctctaacatggggtgcactcttattcttgaggattccaggaataactttttaattcagatgtttcattacattaagacttctctggctccactaccctgctacgtttatctgattgagcatccttccctcaaatatgcgaccactcgttccatagccaagtatcctgtgggtatagaagttggtcctcagcctcaaggggttctgagagctgatatcttggatcaaatgagaaaaatgattaaacatgctcttgattttatacatcatttcaatgaaggaaaagaatttcctccctgcgccattgaggtctataaaattatagagaaagttgattacccccgggatgaaaatggagaaattgctgctatcatccatcctaatctgcaggatcaagactggaaaccactgcatcctggggatcccatgtttttaactcttgatgggaagacgatcccactgggcggagactgtaccgtgtaccccgtgtttgtgaatgaggccgcatattacgaaaagaaagaagcttttgcaaagacaactaaactaacgctcaatgcaaaaagtattcgctgctgtttacattag; NM 023113. (SEQ ID NO: 101)hexosaminidaseA(HEXA): NM_000520: atgacaagctccaggctttggttttcgctgctgctggcggcagcgttcgcaggacgggcgacggccctctggccctggcctcagaacttccaaacctccgaccagcgctacgtcctttacccgaacaactttcaattccagtacgatgtcagctcggccgcgcagcccggctgctcagtcctcgacgaggccttccagcgctatcgtgacctgcttttcggttccgggtcttggccccgtccttacctcacagggaaacggcatacactggagaagaatgtgttggttgtctctgtagtcacacctggatgtaaccagcttcctactttggagtcagtggagaattataccctgaccataaatgatgaccagtgtttactcctctctgagactgtctggggagctctccgaggtctggagacttttagccagcttgtttggaaatctgctgagggcacattctttatcaacaagactgagattgaggactttccccgctttcctcaccggggcttgctgttggatacatctcgccattacctgccactctctagcatcctggacactctggatgtcatggcgtacaataaattgaacgtgttccactggcatctggtagatgatccttccttcccatatgagagcttcacttttccagagctcatgagaaaggggtcctacaaccctgtcacccacatctacacagcacaggatgtgaaggaggtcattgaatacgcacggctccggggtatccgtgtgcttgcagagtttgacactcctggccacactttgtcctggggaccaggtatccctggattactgactccttgctactctgggtctgagccctctggcacctttggaccagtgaatcccagtctcaataatacctatgagttcatgagcacattcttcttagaagtcagctctgtcttcccagatttttatcttcatcttggaggagatgaggttgatttcacctgctggaagtccaacccagagatccaggactttatgaggaagaaaggcttcggtgaggacttcaagcagctggagtccttctacatccagacgctgctggacatcgtctcttcttatggcaagggctatgtggtgtggcaggaggtgtttgataataaagtaaagattcagccagacacaatcatacaggtgtggcgagaggatattccagtgaactatatgaaggagctggaactggtcaccaaggccggcttccgggcccttctctctgccccctggtacctgaaccgtatatcctatggccctgactggaaggatttctacgtagtggaacccctggcatttgaaggtacccctgagcagaaggctctggtgattggtggagaggcttgtatgtggggagaatatgtggacaacacaaacctggtccccaggctctggcccagagcaggggctgttgccgaaaggctgtggagcaacaagttgacatctgacctgacatttgcctatgaacgtttgtcacacttccgctgtgagttgctgaggcgaggtgtccaggcccaacccctcaatgtaggcttctgtgagcaggagtttgaacagacctga. (SEQ ID NO: 102)Lesch_Nyhan_syndromc(HRPT): NM_000194: atggcgacccgcagccctggcgtcgtgattagtgatgatgaaccaggttatgaccttgatttattttgcatacctaatcattatgctgaggatttggaaagggtgtttattcctcatggactaattatggacaggactgaacgtcttgctcgagatgtgatgaaggagatgggaggccatcacattgtagccctctgtgtgctcaaggggggctataaattctttgctgacctgctggattacatcaaagcactgaatagaaatagtgatagatccattcctatgactgtagattttatcagactgaagagctattgtaatgaccagtcaacaggggacataaaagtaattggtggagatgatctctcaactttaactggaaagaatgtcttgattgtggaagatataattgacactggcaaaacaatgcagactttgctttccttggtcaggcagtataatccaaagatggtcaaggtcgcaagcttgctggtgaaaaggaccccacgaagtgttggatataagccagactttgttggatttgaaattccagacaagtttgttgtaggatatgcccttgactataatgaatacttcagggatttgaatcatgtttgtgtcattagtgaaactggaaaagcaaaatacaaagcctaa; NM_204848. Huntingtin; NM_10414;(SEQ ID NO: 103)GUSB; NM_000181: atggcccgggggtcggcggttgcctgggcggcgctcgggccgttgttgtggggctgcgcgctggggctgcagggcgggatgctgtacccccaggagagcccgtcgcgggagtgcaaggagctggacggcctctggagcttccgcgccgacttctctgacaaccgacgccggggcttcgaggagcagtggtaccggcggccgctgtgggagtcaggccccaccgtggacatgccagttccctccagcttcaatgacatcagccaggactggcgtctgcggcattttgtcggctgggtgtggtacgaacgggaggtgatcctgccggagcgatggacccaggacctgcgcacaagagtggtgctgaggattggcagtgcccattcctatgccatcgtgtgggtgaatggggtcgacacgctagagcatgaggggggctacctccccttcgaggccgacatcagcaacctggtccaggtggggcccctgccctcccggctccgaatcactatcgccatcaacaacacactcacccccaccaccctgccaccagggaccatccaatacctgactgacacctccaagtatcccaagggttactttgtccagaacacatattttgactttttcaactacgctggactgcagcggtctgtacttctgtacacgacacccaccacctacatcgatgacatcaccgtcaccaccagcgtggagcaagacagtgggctggtgaattaccagatctctgtcaagggcagtaacctgttcaagttggaagtgcgtcttttggatgcagaaaacaaagtcgtggcgaatgggactgggacccagggccaacttaaggtgccaggtgtcagcctctggtggccgtacctgatgcacgaacgccctgcctatctgtattcattggaggtgcagctgactgcacagacgtcactggggcctgtgtctgacttctacacactccctgtggggatccgcactgtggctgtcaccaagagccagttcctcatcaatgggaaacctttctatttccacggtgtcaacaagcatgaggatgcggacatccgagggaagggcttcgactggccgctgctggtgaaggacttcaacctgcttcgctggcttggtgccaacgctttccgtaccagccactacccctatgcagaggaagtgatgcagatgtgtgaccgctatgggattgtggtcatcgatgagtgtcccggcgtgggcctggcgctgccgcagttcttcaacaacgtttctctgcatcaccacatgcaggtgatggaagaagtggtgcgtagggacaagaaccaccccgcggtcgtgatgtggtctgtggccaacgagcctgcgtcccacctagaatctgctggctactacttgaagatggtgatcgctcacaccaaatccttggacccctcccggcctgtgacctttgtgagcaactctaactatgcagcagacaagggggctccgtatgtggatgtgatctgtttgaacagctactactcttggtatcacgactacgggcacctggagttgattcagctgcagctggccacccagtttgagaactggtataagaagtatcagaagcccattattcagagcgagtatggagcagaaacgattgcagggtttcaccaggatccacctctgatgttcactgaagagtaccagaaaagtctgctagagcagtaccatctgggtctggatcaaaaacgcagaaaatacgtggttggagagctcatttggaattttgccgatttcatgactgaacagtcaccgacgagagtgctggggaataaaaaggggatcttcactcggcagagacaaccaaaaagtgcagcgttccttttgcgagagagatactggaagattgccaatgaaaccaggtatccccactcagtagccaagtcacaatgtttggaaaacagcccgtttacttga; NM_010368. NPC1: NMJ)00271;NM_006432.(SEQ ID NO: 104)hexosaminidaseB: NM_000521; atggagctgtgcgggctggggctgccccggccgcccatgctgctggcgctgctgttggcgacactgctggcggcgatgttggcgctgctgactcaggtggcgctggtggtgcaggtggcggaggcggctcgggccccgagcgtctcggccaagccggggccggcgctgtggcccctgccgctctcggtgaagatgaccccgaacctgctgcatctcgccccggagaacttctacatcagccacagccccaattccacggcgggcccctcctgcaccctgctggaggaagcgtttcgacgatatcatggctatatttttggtttctacaagtggcatcatgaacctgctgaattccaggctaaaacccaggttcagcaacttcttgtctcaatcacccttcagtcagagtgtgatgctttccccaacatatcttcagatgagtcttatactttacttgtgaaagaaccagtggctgtccttaaggccaacagagtttggggagcattacgaggtttagagacctttagccagttagtttatcaagattcttatggaactttcaccatcaatgaatccaccattattgattctccaaggttttctcacagaggaattttgattgatacatccagacattatctgccagttaagattattcttaaaactctggatgccatggcttttaataagtttaatgttcttcactggcacatagttgatgaccagtctttcccatatcagagcatcacttttcctgagttaagcaataaaggaagctattctttgtctcatgtttatacaccaaatgatgtccgtatggtgattgaatatgccagattacgaggaattcgagtcctgccagaatttgatacccctgggcatacactatcttggggaaaaggtcagaaagacctcctgactccatgttacagtagacaaaacaagttggactcttttggacctataaaccctactctgaatacaacatacagcttccttactacatttttcaaagaaattagtgaggtgtttccagatcaattcattcatttgggaggagatgaagtggaatttaaatgttgggaatcaaatccaaaaattcaagatttcatgaggcaaaaaggctttggcacagattttaagaaactagaatctttctacattcaaaaggttttggatattattgcaaccataaacaagggatccattgtctggcaggaggtttttgatgataaagcaaagcttgcgccgggcacaatagttgaagtatggaaagacagcgcatatcctgaggaactcagtagagtcacagcatctggcttccctgtaatcctttctgctccttggtacttagatttgattagctatggacaagattggaggaaatactataaagtggaacctcttgattttggcggtactcagaaacagaaacaacttttcattggtggagaagcttgtctatggggagaatatgtggatgcaactaacctcactccaagattatggcctcgggcaagtgctgttggtgagagactctggagttccaaagatgtcagagatatggatgacgcctatgacagactgacaaggcaccgctgcaggatggtcgaacgtggaatagctgcacaacctctttatgctggatattgtaaccatgagaacatgtaa. (SEQ ID NO: 105)galactosidase, alpha(GLA): NM000169:atgcagctgaggaacccagaactacatctgggctgcgcgcttgcgcttcgcttcctggccctcgtttcctgggacatccctggggctagagcactggacaatggattggcaaggacgcctaccatgggctggctgcactgggagcgcttcatgtgcaaccttgactgccaggaagagccagattcctgcatcagtgagaagctcttcatggagatggcagagctcatggtctcagaaggctggaaggatgcaggttatgagtacctctgcattgatgactgttggatggctccccaaagagattcagaaggcagacttcaggcagaccctcagcgctttcctcatgggattcgccagctagctaattatgttcacagcaaaggactgaagctagggatttatgcagatgttggaaataaaacctgcgcaggcttccctgggagttttggatactacgacattgatgcccagacctttgctgactggggagtagatctgctaaaatttgatggttgttactgtgacagtttggaaaatttggcagatggttataagcacatgtccttggccctgaataggactggcagaagcattgtgtactcctgtgagtggcctctttatatgtggccctttcaaaagcccaattatacagaaatccgacagtactgcaatcactggcgaaattttgctgacattgatgattcctggaaaagtataaagagtatcttggactggacatcttttaaccaggagagaattgttgatgttgctggaccagggggttggaatgacccagatatgttagtgattggcaactttggcctcagctggaatcagcaagtaactcagatggccctctgggctatcatggctgctcctttattcatgtctaatgacctccgacacatcagccctcaagccaaagctctccttcaggataaggacgtaattgccatcaatcaggaccccttgggcaagcaagggtaccagcttagacagggagacaactttgaagtgtgggaacgacctctctcaggcttagcctgggctgtagctatgataaaceggcaggagattggtggacctcgctcttataccatcgcagttgcttccctgggtaaaggagtggcctgtaatcctgcctgcttcatcacacagctcctccctgtgaaaaggaagctagggttctatgaatggacttcaaggttaagaagtcacataaatcccacaggcactgttttgcttcagctagaaaatacaatgcagatgtcattaaaagacttactttaa (SEQ ID NO: 106)glucosidasE beta_acid(GBA): NM_000157:atggagttttcaagtccttccagagaggaatgtcccaagcctttgagtagggtaagcatcatggctggcagcctcacaggattgcttctacttcaggcagtgtcgtgggcatcaggtgcccgcccctgcatccctaaaagcttcggctacagctcggtggtgtgtgtctgcaatgccacatactgtgactcctttgaccccccgacctttcctgcccttggtaccttcagccgctatgagagtacacgcagtgggcgacggatggagctgagtatggggcccatccaggctaatcacacgggcacaggcctgctactgaccctgcagccagaacagaagttccagaaagtgaagggatttggaggggccatgacagatgctgctgctctcaacatccttgccctgtcaccccctgcccaaaatttgctacttaaatcgtacttctctgaagaaggaatcggatataacatcatccgggtacccatggccagctgtgacttctccatccgcacctacacctatgcagacacccctgatgatttccagttgcacaacttcagcctcccagaggaagataccaagctcaagatacccctgattcaccgagccctgcagttggcccagcgtcccgtttcactccttgccagcccctggacatcacccacttggctcaagaccaatggagcggtgaatgggaaggggtcactcaagggacagcccggagacatctaccaccagacctgggccagatactttgtgaagttcctggatgcctatgctgagcacaagttacagttctgggcagtgacagctgaaaatgagccttctgctgggctgttgagtggataccccttccagtgcctgggcttcacccctgaacatcagcgagacttcattgcccgtgacctaggtcctaccctcgccaacagtactcaccacaatgtccgcctactcatgctggatgaccaacgcttgctgctgccccactgggcaaaggtggtactgacagacccagaagcagctaaatatgttcatggcattgctgtacattggtacctggactttctggctccagccaaagccaccctaggggagacacaccgcctgttccccaacaccatgctctttgcctcagaggcctgtgtgggctccaagttctgggagcagagtgtgcggctaggctcctgggatcgagggatgcagtacagccacagcatcatcacgaacctcctgtaccatgtggtcggctggaccgactggaaccttgccctgaaccccgaaggaggacccaattgggtgcgtaactttgtcgacagtcccatcattgtagacatcaccaaggacacgttttacaaacagcccatgttctaccaccttggccacttcagcaagttcattcctgagggctcccagagagtggggctggttgccagtcagaagaacgacctggacgcagtggcactgatgcatcccgatggctctgctgttgtggtcgtgctaaaccgctcctctaaggatgtgcctcttaccatcaaggatcctgctgtgggcttcctggagacaatctcacctggctactccattcacacctacctgtggcgtcgccagtga; NM_008094.(SEQ ID NO: 107) von_Hippel_Lindau_tumor_suppressor(VHL): NM_000551:atgccccggagggcggagaactgggacgaggccgaggtaggcgcggaggaggcaggcgtcgaagagtacggccctgaagaagacggcggggaggagtcgggcgccgaggagtccggcccggaagagtccggcccggaggaactgggcgccgaggaggagatggaggccgggcggccgcggcccgtgctgcgctcggtgaactcgcgcgagccctcccaggtcatcttctgcaatcgcagtccgcgcgtcgtgctgcccgtatggctcaacttcgacggcgagccgcagccctacccaacgctgccgcctggcacgggccgccgcatccacagctaccgaggtcacctttggctcttcagagatgcagggacacacgatgggcttctggttaaccaaactgaattatttgtgccatctctcaatgttgacggacagcctatttttgccaatatcacactgccagtgtatactctgaaagagcgatgcctccaggttgtccggagcctagtcaagcctgagaattacaggagactggacatcgtcaggtcgctctacgaagatctggaagaccacccaaatgtgcagaaagacctggagcggctgacacaggagcgcattgcacatcaacggatgggagattga. (SEQ ID NO: 108)Beta_globin(HBB): NM_000518: atggtgcatctgactcctgaggagaagtctgccgttactgccctgtggggcaaggtgaacgtggatgaagttggtggtgaggccctgggcaggctgctggtggtctacccttggacccagaggttctttgagtcctttggggatctgtccactcctgatgctgttatgggcaaccctaaggtgaaggctcatggcaagaaagtgctcggtgcctttagtgatggcctggctcacctggacaacctcaagggcacctttgccacactgagtgagctgcactgtgacaagctgcacgtggatcctgagaacttcaggctcctgggcaacgtgctggtctgtgtgctggcccatcactttggcaaagaattcaccccaccagtgcaggctgcctatcagaaagtggtggctggtgtggctaatgccctggcccacaagtatcactaa.(SEQ ID NO: 109)PARK2: NM_013988: atgatagtgtttgtcaggttcaactccagccatggtttcccagtggaggtcgattctgacaccagcatcttccagctcaaggaggtggttgctaagcgacagggggttccggctgaccagttgcgtgtgattttcgcagggaaggagctgaggaatgactggactgtgcaggaatttttctttaaatgtggagcacaccccacctctgacaaggaaacatcagtagctttgcacctgatcgcaacaaatagtcggaacatcacttgcattacgtgcacagacgtcaggagccccgtcctggttttccagtgcaactcccgccacgtgatttgcttagactgtttccacttatactgtgtgacaagactcaatgatcggcagtttgttcacgaccctcaacttggctactccctgccttgtgtggctggctgtcccaactccttgattaaagagctccatcacttcaggattctgggagaagagcagtacaaccggtaccagcagtatggtgcagaggagtgtgtcctgcagatggggggcgtgttatgcccccgccctggctgtggagcggggctgctgccggagcctgaccagaggaaagtcacctgcgaagggggcaatggcctgggctgtgggtttgccttctgccgggaatgtaaagaagcgtaccatgaaggggagtgcagtgccgtatttgaagcctcaggaacaactactcaggcctacagagtcgatgaaagagccgccgagcaggctcgttgggaagcagcctccaaagaaaccatcaagaaaaccaccaagccctgtccccgctgccatgtaccagtggaaaaaaatggaggctgcatgcacatgaagtgtccgcagccccagtgcaggctcgagtggtgctggaactgtggctgcgagtggaaccgcgtctgcatgggggaccactggttcgacgtgtag;NM 004562; NM 020093.

The contents of all parenthetically cited publications and the followingUnited States Patents, are noted and incorporated by reference in theirentireties: U.S. Pat. Nos. 7,211,247, 5,677,139, 6,432,711 and5,453,357, 5,593,875, 5,783,566, 5,928,944, 5,910,488, 5,824,547.

What is claimed are:
 1. A cell culture dish made of non media-permeablematerial and having a base and a plurality of separate juxtaposedside-by-side wells having common interior well walls preventing physicalcontact or movement of chemical factors between the separated cell ortissue cultures, the walls having different heights and defining anouter “surround” cell or tissue culture and one or more inner “center”cell or tissue cultures to enable contained cell or tissue communicationbetween the well spaces, the wells configured to comprise two or morecell or tissue cultures, the wells permitting signal communicationbetween the cells or tissues situated within said wells, and furthercomprising wall material, electrodes and/or electrode contacts, and welldimensions suitable for facilitating electroporation.
 2. The methodinducing cellular behavior displayed in a first cell population that isdisplayed in a second cell population wherein the method comprisesculturing the cells in the cell culture dish of claim
 1. 3. The methodof claim 2 where the desired induced behavior comprises one or morechanges in signal transduction.
 4. The method of claim 2 where thedesired induced behavior comprises one or more changes in ion currentflows.
 5. The method of claim 2 where the desired induced behaviorcomprises a change in gene expression.
 6. The method of claim 2 wherethe desired induced behavior comprises a change in protein expression.7. The method of claim 2 where the desired induced behavior comprises achange in cellular markers.
 8. The method of claim 2 where the desiredinduced behavior comprises a change in cellular phenotype.
 9. The methodof claim 2 where the desired induced behavior comprises a change inantibody reactivity.
 10. The method of claim 2 where the desired inducedbehavior comprises a change allowing differential cell sorting.
 11. Themethod of claim 2 where the desired induced behavior comprises a changein potency.
 12. The method of claim 2 wherein cells of the first cellpopulation are induced to greater potency.
 13. The method of claim 2wherein cells of the first cell population are induced to a new,differentiated cell phenotype.
 14. The method of claim 2 wherein cellsof the first cell population are further cultured in a 2 Dimensional or3 Dimensional format.
 15. The method of claim 2 wherein cells of thefirst cell population are genetically-modified.
 16. The method of claim2 wherein cells of the first cell population are contacted with agentsfurther promoting the desired induced behavior.
 17. The method of claim2 wherein the first and/or second cell population is a somatic cellpopulation.
 18. The method of claim 2 wherein the first and/or secondcell population is stem cell population.
 19. The method of claim 2.wherein the first and/or second cell population displays multipotent,pluripotent or totipotent stem cell behavior.
 20. The method of claim 2wherein the medium comprises agents promoting cellular differentiation.21. The method of claim 2 wherein the medium comprises nucleic acids orproteins.
 22. The method of claim 2 wherein the medium comprisesnon-nucleic acids and non-proteins.
 23. The method of claim 21 whereincells of the first cell population are contacted with nucleic acid orprotein transcription factors or other cell fate determinants.
 24. Thecells, vectors, agents, proteins, nucleic acids, transcription factors,and other cell fate determinants of claims 1-23.
 25. The method oftreatment comprising administering to a subject, the cells, agents,nucleic acids or proteins of claims 1-24.